Mature Dendritic Cell Compositions and Methods for Culturing Same

ABSTRACT

This invention provides methods to prepare and use immunostimulatory cells for enhancing an immune response. The invention provides a method for preparing mature dendritic cells (DCs), comprising the sequential steps of: (a) signaling isolated immature dendritic cells (iDCs) with a first signal comprising an interferon gamma receptor (IFN-γR) agonist and/or a tumor necrosis factor alpha receptor (TNF-αR) agonist to produce signaled dendritic cells; and (b) signaling said signaled dendritic cells with a second transient signal comprising an effective amount of a CD40 agonist to produce CCR7 +  mature dendritic cells. Also provided by this invention are enriched populations of dendritic cells prepared by the methods of the invention. Such dendritic cells have enhanced immunostimulatory properties and increased IL-12 secretion and/or decreased IL-10 secretion. CD40 signaling can be initiated by one or more of polypeptide translated from an exogenous polynucleotide encoding CD40L (e.g., mRNA or DNA), an agonistic antibody to CD40 receptor or by CD40 ligand polypeptide. The enriched populations can be further modified by the administration of an immunogen to the DC. The DC will take up and process the immunogen on its cell surface.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/400,774, filed 7 Apr. 2006, which is a continuation-in-part of U.S.application Ser. No. 11/246,387, filed 7 Oct. 2005, and is also acontinuation-in-part of PCT application PCT/US2005/036304, filed 7 Oct.2005, both of which claim the benefit of U.S. Provisional Application60/522,512, filed 7 Oct. 2004, the contents of each of which are herebyincorporated by reference in their entireties.

FIELD OF THE INVENTION

The present invention relates to the generation of mature dendriticcells and their use in cell therapy and to educate immune effectorcells. The mature dendritic cells can be generated from immaturedendritic cells.

BACKGROUND

Cell therapy utilizes modified antigen presenting cells (APCs) or immuneeffector cells to initiate an immune response in a patient. Antigenpresenting cells are central to cell therapy because they initiate theimmune response. Indeed, they are the only cells capable of inducing aprimary immune response from the T lymphocytes.

Dendritic cells (DC) are the most potent APCs involved in adaptiveimmunity. They coordinate the initiation of immune responses by naive Tcells and B cells and induce antigen-specific cytotoxic T lymphocyte(CTL) responses. DCs are specialized in several ways to prime helper andkiller T cells in vivo. For example, immature DCs that reside inperipheral tissues are equipped to capture antigens and to produceimmunogenic MHC-peptide complexes. In response to maturation-inducingstimuli such as inflammatory cytokines, immature DCs develop into potentT cell stimulators by upregulating adhesion and costimulatory molecules.At the same time, they migrate into secondary lymphoid organs to selectand stimulate rare antigen-specific T cells. However, potent stimulationof T cells occurs only after DC maturation, a process that increases theavailability of MHC/peptide complexes on the cell surface, in additionto co-stimulatory molecules, that direct the effector function of theresponding T-cells. Indeed, immature DCs may be harmful in anti-tumorand other immunotherapies because they can induce immunotolerance ratherthan immunostimulation.

Co-stimulation is typically necessary for a T cell to produce sufficientcytokine levels that induce clonal expansion. One characteristic ofdendritic cells which makes them potent antigen presenting cells is thatthey are rich in co-stimulatory molecules of the immune response, suchas the molecules CD80 and CD86, which activate the molecule CD28, on Tlymphocytes. In return, T-helper cells express CD40L, which ligates CD40on DCs. These mutual interactions between DCs and T-cells leads to‘maturation’ of the former, and the development of effector function inthe latter. The expression of adhesion molecules, like the molecule CD54or the molecule CD11a/CD18, facilitate the co-operation between thedendritic cells and the T-cells. Another special characteristic ofdendritic cells is to deploy different functions depending on theirstage of differentiation. Thus, the capture of the antigen and itstransformation are the two principal functions of the immature dendriticcell, whereas its capacities to present the antigen in order tostimulate the T cells increases as the dendritic cells migrate into thetissues and the lymphatic ganglia. This change of functionalitycorresponds to a maturation of the dendritic cell. Thus, the passage ofthe immature dendritic cell to the mature dendritic cell represents afundamental step in the initiation of the immune response.Traditionally, this maturation was followed by monitoring the change ofthe surface markers on the DCs during this process. Some of the moreimportant cell surface markers characteristic of the different stages ofmaturation of the dendritic cells are summarized in Table I, below.However, the surface markers can vary depending upon the maturationprocess.

TABLE I Cell type Surface markers Hematopoietic stem CD34+ cellMonocytes CD14++, DR+, CD86+, CD16+/−, CD54+, CD40+ Immature dendriticCD14+/−, CD16−, CD80+/−, CD83−, CD86+, cell CD1a+, CD54+, DQ+, DR++Mature dendritic CD14−, CD83++, CD86++, CD80++, DR+++, cell DQ++,CD40++, CD54++, CD1a+/−

Mature DCs are currently preferred to immature DCs for immunotherapy.Only fully mature DC progeny lack GM-CSF Receptor (GM-CSF-R) and remainstablely mature upon removal/in the absence of GM-CSF. Also, mature DCshave been shown to be superior in inducing T cell responses in vitro andin vivo. In contrast, immature DCs are reported to induce tolerance invitro (Jonuleit et al. (2000) Exp. Med. 192:1213) as well as in vivo(Dhodapkar et al. (2001) Exp. Med. 193:233) by inducing regulatory Tcells. Mature dendritic cells also are useful to take up and presentantigen to T-lymphocytes in vitro or in vivo. The modified, antigenpresenting DCs and/or T cells educated from these modified DCs have manyapplications, including diagnostic, therapy, vaccination, research,screening and gene delivery.

It is difficult to isolate mature dendritic cells from peripheral bloodbecause less than 1% of the white blood cells belongs to this category.Mature DCs are also difficult to extract from tissues. This difficulty,in combination with the potential therapeutic benefit of DCs in celltherapy, has driven research and development toward new methods togenerate mature dendritic cells using alternative sources. Severalmethods are reported to produce mature DCs from immature dendriticcells.

For example, Jonuleit et al. (Eur J Immunol (1997) 12:3135-3142)disclose maturation of immature human DCs by culture in mediumcontaining a cytokine cocktail (IL-1β, TNF-α, IL-6 and PGE₂).

WO 95/28479 discloses a process for preparing dendritic cells byisolating peripheral blood cells and enriching for CD34⁺ blood precursorcells, followed by expansion with a combination of hematopoietic growthfactors and cytokines.

European Patent Publication EP-A-0 922 758 discloses the production ofmature dendritic cells from immature dendritic cells derived frompluripotential cells having the potential of expressing eithermacrophage or dendritic cell characteristics. The method requirescontacting the immature dendritic cells with a dendritic cell maturationfactor containing IFN-γ.

European Patent Publication EP-B-0 633930 teaches the production ofhuman dendritic cells by first culturing human CD34⁺ hematopoietic cells(i) with GM-CSF, (ii) with TNF-α and IL-3, or (iii) with GM-CSF andTNF-α to induce the formation of CD1a⁺ hematopoietic cells.

Patent Publication No. 2004/0152191 discloses the maturation ofdendritic cells by contacting them with RU 41740.

U.S. Patent Publication No. 2004/0146492 teaches a process for producingrecombinant dendritic cells by transforming hematopoietic stem cellsfollowed by differentiation of the stem cells into dendritic cells byculture in medium containing GM-CSF.

U.S. Patent Publication No. 2004/0038398 discloses methods for thepreparation of substantially purified populations of DCs and monocytesfrom the peripheral blood of mammals. Myeloid cells are isolated fromthe mammal and DCs are separated from this population to yield anisolated subpopulation of monocytes. DCs are then enriched by negativeselection with anti-CD2 antibodies to remove T cells.

Although mature DCs are functionally competent and are therefore usefulto induce antigen-specific T cells, not all mature DCs are optimized toinduce these responses. It has been shown that some mature DCs may alsostimulate T helper cells by secreting IL-12. Macatonia et al. (1995)Immunol. 154:507 1; Ahuja et al. (1998) Immunol. 161:868 and Unintfordet al. (1999) Immunol. 97:588. IL-12 also has been shown to enhanceantigen-specific CD8+ T cell response to antigen in an animal model.Schmidt et al. (1999) Immunol. 163:2561.

Mosca et al. (2000) Blood 96:3499, disclose that culture of DC in AIM Vmedium containing both soluble CD40L trimer and IFNγ1b results inincreased IL-12 expression in comparison to culture in medium containingonly soluble CD40L trimer.

Koya et al. (2003) J. Immunother. 26(5): 451 report that IL-12expression can be enhanced by tranducing immature DCs, in the presenceof IFNγ, with a lentiviral vector encoding CD40 Ligand. Greater than 90%of the CD40L transduced DCs expressed CD83 on their cell surface.Unfortuantely, lentiviral transduced cells are not suitable fortherapeutic purposes, and proviral integration into the genome of thetransduced cell can result in leukemia. Furthermore, persistantexpression of CD40L may have detrimental effects on APC function andviability.

This work supplemented the earlier work of Mackey, et al. (1998) J.Immunol. 161:2094 who reported that in vivo, DCs require maturation viaCD40 to generate anti-tumor immunity. Similarly, Kuniyoshi, J. S. et al.(1999) Cell Immunol. 193:48 have shown that DCs treated with solubletrimeric CD40 Ligand plus IFN-γ stimulated potent T-cell proliferationand induced T cells with augmented antigen-specific lysis. Kalady, M. F.et al. (2004) J. Surg. Res. 116:24, reported that human monocyte derivedDCs transfected with mRNA encoding melanoma antigen MART-1 or influenzaM1 matrix protein exposed to different maturation stimuli added eithersimultaneously or sequentially showed variability in antigenpresentation, IL-12 secretion and immunogenicity of effector T cellsraised in the presence of these DC's. Most importantly, this studyshowed that the application of a ‘cytokine cocktail’ consisting ofIL-1β, TNF-α, IL-6 and PGE₂, followed by extracellular soluble CD40Lprotein was superior to applying all the agents simultaneously. However,these authors did not study the combination of IFN-γ signaling withtransient CD40L signalling in a sequential process. Moreover, despitethe production of IL-12 when IFN-γ and CD40L are concomitantly added tothe culture medium, the recent prior art shows that the resulting DCsare actually immunosuppressive, rather than pro-inflammatory (Hwu et al.(2000) J. Immunol. 164: 3596 ; Munn et al. (2002) 297:1867 ; andGrohmann et al. (2003) Trends Immunol. 24:242) due to the induction ofan enzyme that metabolized tryptophan resulting in the starvation ofresponder T-cells that then fail to proliferate. Thus, currentliterature suggests that the combination of IFN-γ and CD40L should notincrease immunopotency. The present invention addresses the long-feltneed to provide improved methods for DC maturation and mature DCs withenhanced immunopotentcy.

SUMMARY OF THE INVENTION

Applicants have discovered that potent immunostimulation occurs whenimmature dendritic cells are sequentially signaled with a first signalcomprising an interferon gamma receptor (IFN-γR) agonist followed by asecond signal comprising a CD40 agonist. Accordingly, this inventionprovides a method for preparing mature dendritic cells (DCs), comprisingthe sequential steps of: (a) signaling isolated immature dendritic cells(iDCs) with a first signal comprising an interferon gamma receptor(IFN-γR) agonist, and optionally a TNF-αR agonist, to produce signaleddendritic cells; and (b) signaling said signaled dendritic cells with asecond transient signal comprising an effective amount of a CD40 agonistto produce CCR7⁺ mature dendritic cells.

In preferred embodiments, the immature DCs are further contacted withPGE₂ and optionally with TNF-α. In alternative embodiments the methodfurther comprises contacting the immature DCs, signaled DCs and/or CCR7⁺mature dendritic cells with a compound selected from the groupconsisting of: galactosylceramides, glycosylceramides,galactofuranosylceramides, arabinopyranosylceramides,α-C-galactosylceramides and α-S-galactosylceramides. Preferably thecompound is a galactosylceramide. Most preferably, thegalactosylceramide is (2S, 3S,4R)-1-O—(alpha-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol(KRN7000).

In another embodiment of the invention, the IFN-γR agonist can bereplaced by a tumor necrosis factor alpha receptor (TNF-αR) agonist.Thus, the invention provides a method for preparing an enrichedpopulation of mature dendritic cells (DCs), comprising sequentiallysignaling immature dendritic cells with a first signal comprising atumor necrosis factor alpha receptor (TNF-αR) agonist followed by asecond signal comprising a CD40 agonist, thereby preparing an enrichedpopulation of mature dendritic cells, wherein said signaling is in theabsence of an effective amount of IL-1β or IL-6. Preferably, theimmature DCs are further contacted with PGE₂.

Preferred IFN-γR agonists are mammalian IFN-γ, preferably human IFN-γand active fragments thereof. Preferred TNF-αR agonists are mammalianTNF-α, preferably human TNF-α and active fragments thereof. PreferredCD40 agonists are mammalian CD40 Ligands (CD40L), preferably human CD40Land active fragments and variants thereof, as well as agonisticantibodies to CD40 receptor. Most preferably, the CD40L agonist is anovel CD40L polypeptide provided herein, consisting of or consistingessentially of amino acid residues 21-261 of SEQ ID NO: 2. Nucleic acidsencoding the novel CD40L polypeptide, transfected DCs and relatedvaccines are also provided.

Signaling can be initiated by providing the signaling agonist in theculture medium, introduction of the agonist into the cell, and/or upontranslation within the dendritic cell of an mRNA encoding an agonisticpolypeptide. The method can be practiced in vivo or ex vivo. Dendriticcells matured ex vivo according to the methods of the invention can thenbe administered to the subject to induce or enhance an immune response.

Each of the dendritic cells can be further modified by theadministration of an immunogen to the DC. The DC will take up andprocess the immunogen, and display it on its cell surface. The immunogencan be delivered in vivo or ex vivo. The matured, cultured DCs can beadministered to a subject to induce or enhance an immune response. Inyet a further embodiment, the antigen loaded mature DCs are used toeducate naïve immune effector cells.

In another aspect, the invention provides a composition comprising invitro matured dendritic cells, such as CD83⁺ CCR7 ⁻ mature DCs and CD83⁺CCR7⁺ mature DCs. Mature dendritic cells of the invention expressincreased levels of IL-12 in comparison to immature dendritic cells,and/or express less than 500 pg IL-10 per million dendritic cells.

In another embodiment, the invention provides a dendritic cell whichpreferentially induces a population of CD28⁺ CD45RA⁻ memory/effector Tcells from a population of antigen-specific T cells. The compositions ofthe invention are useful to raise an immune response in a subject byadministering to the subject an effective amount of the population.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NO: 1 is a human CD40L cDNA. Nucleotides 40 to 825 represent thecoding region, including the ATG translation start codon and the TGAtranslational stop codon.

SEQ ID NO: 2 is an amino acid sequence for full length human CD40Lprotein.

SEQ ID NO: 3 is a human CD40 cDNA. Nucleotides 67 to 522 represent thecoding region, including the ATG translation start codon and the TAGtranslational stop codon.

SEQ ID NO: 4 is an amino acid sequence for human CD40 (the receptor forCD40L).

SEQ ID NO: 5 is a human IFN-γ cDNA. Nucleotides 109 to 609 represent thecoding region, including the ATG translation start codon and thetranslational stop codon.

SEQ ID NO: 6 is an amino acid sequence for human IFN-γ.

SEQ ID NO: 7 is a human TNF-α cDNA. Nucleotides 170 to 971 represent thecoding region, including the ATG translation start codon and the TGAtranslational stop codon.

SEQ ID NO: 8 is an amino acid sequence for human TNF-α.

SEQ ID NO: 9 is a mouse CD40L cDNA. Nucleotides 13 to 795 represent thecoding region, including the ATG translation start codon and the TGAtranslational stop codon.

SEQ ID NO: 10 is an amino acid sequence for full length mouse CD40Lprotein.

SEQ ID NO: 11 is a CD40L 5′ primer.

SEQ ID NO: 12 is a CD40L 3′ primer.

SEQ ID NO: 13 is the DNA sequence corresponding to an optimized humanCD40L mRNA.

SEQ ID NO: 14 is the CD40 Receptor 3′UTR.

SEQ ID NO: 15 is the untranslated region of final exon of the humanbeta-actin 3′ UTR.

SEQ ID NO: 16 is the minimal functional element of the human beta-actin3′ UTR.

SEQ ID NO: 17 is the simian rotavirus Gene 6 3′UTR.

SEQ ID NO: 18 is the minimal functional element of the simian rotavirusGene 6 3′UTR.

SEQ ID NO: 19 is the human Hsp70 5 ′UTR (HSPA1A).

SEQ ID NO: 20 is the mouse VEGF 5′UTR.

SEQ ID NO: 21 is the minimal functional element of the mouse VEGF 5′UTR.

SEQ ID NO: 22 is the Spleen Necrosis Virus LTR RU5 Region.

SEQ ID NO: 23 is the Tobacco Etch Virus 5′ Leader sequence.

SEQ ID NOs: 24-26 are HLA-A201 restricted MART-APL peptide, nativepeptide and PSA-1 peptide, respectively.

SEQ ID NO: 27 is the human α-globin 3′UTR.

SEQ ID NO: 28 is the human β-globin 3′UTR.

SEQ ID NO: 29 is the human β-globin 3′UTR, minus Purine-Rich Element 3.

SEQ ID NO: 30 shows the cDNA sequence corresponding to the CD40L RNAtranscribed from the ΔXE-met#1 plasmid, prior to polyadenylation.

SEQ ID NO: 31 shows the sequence of the CD40L polypeptide translatedfrom the the RNA of SEQ ID NO: 30, and is equivalent to amino acidresidues 21-261 of SEQ ID NO: 2.

SEQ ID NO: 32 shows the cDNA sequence corresponding to the RNAtranscribed from the CD40L ΔXE+rotoavirus gene 6 3′UTR plasmid.

SEQ ID NO: 33 shows the cDNA sequence corresponding to the RNAtranscribed from the CD40L ΔXE-met#1+rotoavirus gene 6 3′UTR plasmid.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows that sequential maturation of DCs with IFN-γ then solubleCD40L results in optimal IL-12p70 secretion. DCs were matured withcytokine cocktail, soluble CD40L alone, or with soluble CD40L plusIFN-γ. Pre-incubation of immature DCs with 1000 U/ml of IFN-γ for 18hrs, followed by addition of soluble CD40L for a further 18 hrs resultsin maximum IL-12p70 release. Applying soluble CD40L first, followed byIFN-γ is perceived as a negative signal, with minimal IL-12p70 release,accompanied by IL-10.

FIG. 2 shows that HELA cells transfected with mRNA encoding CD40L andhaving a polyA tail of >100 nucleotides express cell surface protein, asdefined by FACS analysis with anti-CD40L (CD 154) antibody.

FIG. 3 shows that IL-12p70 secretion from CD40L mRNA transfected cellsis proportional to the size of the transfection payload. DCs weretransfected with a titration of CD40L mRNA followed immediately by theaddition of 1000 U/ml IFN-γ. At least 4 μg per million DCs of CD40L mRNAis required to induce significant levels of IL-12p70 release.

FIG. 4 shows that at least 100 U/ml of IFN-γ is required to synergizewith the CD40L mRNA payload to induce maximal IL-12p70 secretion. DCswere transfected with 4 μg CD40L mRNA per million cells, and immediatelyincubated with a titration of IFN-γ. IL-12p70 and IL-10 were measured inculture supernatants after 24 hrs.

FIG. 5A shows that IL-12p70 secretion induced by CD40L/IFN-γ occursapproximately 24 hrs after transfection of DCs and culture in thepresence of IFN-γ. DCs were transfected with 4 μg CD40L mRNA per millioncells, and immediately cultured with 1000 U/ml IFN-γ. Supernatants werecollected from replica cultures at the designated times, and assayed forIL-12p70 and IL-10 content.

FIG. 5B shows that addition of TNF-α to CD40L mRNA transfected DCsresults in the generation of IL-12p70, but the level of expression isless than that achieved with IFN-γ as the co-maturation agent.

FIG. 5C shows that the use of TNF-α as the co-maturation factor alsoresults in elevated levels of IL-10 compared to the use of IFN-γ.

FIGS. 6A and 6B show that DCS transfected with mRNA encoding CD40Ldemonstrate cellular expression as defined by FACS analysis withanti-CD40L (CD154) antibody. In FIG. 6A, DCs were transfected with 4 μgCD40L mRNA per million cells and analyzed at various time points. Themajority of CD40L is localized within an intracellular compartment asdemonstrated by a 4 hour time point where surface expression isconsiderably lower. FIG. 6B shows that significant intracellularexpression is evident at 60 minutes with 27% positive DCs and increasingto 79% by 3 hours.

FIG. 6C shows transient expression of CD40L protein post transfection ofDC with CD40L encoding mRNA.

FIG. 7 shows that DCs transfected with CD40L mRNA and cultured in thepresence of IFN-γ secrete IL-12p70 despite the presence of an excess ofblocking anti-CD40L antibody, CD40/CD40L interactions operate within an“intracellular” compartment. DCs were transfected 4 μg CD40L mRNA andimmediately cultured with 1000 U/ml IFN-γ in the presence of either 10or 50 μg/ml of blocking anti-CD40L antibody. IL-12 p70 release isreduced by only 50%, indicating that intracellular signaling, ratherthan cell to cell signaling is the primary pathway for the induction ofIL-12p70.

FIG. 8 shows that DCs transfected with CD40L mRNA and co-cultured withIFN-γ require the presence of PGE₂ to enable chemokine dependentmigration. DCs were transfected with a titration of CD40L mRNA andimmediately incubated with 1000 U/ml IFN-γ and 1 μg/ml PGE₂. DCstransfected with eGFP and matured with a cytokine cocktail containingPGE₂ represent a positive control. After 18 hrs of maturation, DCs fromeach culture condition were tested in “transwell” migration assaysagainst the lymph node homing chemokines, CCL19 and 21. DC migration wasproportional to the size of the CD40L mRNA payload.

FIG. 9 shows that DCs matured via transfection with CD40L mRNA andcultured in the presence of IFN-γ and PGE₂ invoke efficient T-cell“recall responses” when compared to DCs matured in the presence of the“cytokine cocktail”. DCs were co-transfected with 2 μg flu M1 mRNA permillion cells as antigen payload, and 4 μg eGFP mRNA control, andsubsequently matured with cytokine cocktail. Alternatively, DCs wereco-transfected with 2 μg flu M1 mRNA per million cells as antigenpayload, and 4 μg CD40L mRNA as the maturation payload. These lattercells were immediately cultured in 1000 U/ml IFN-γ and 1 μg/ml PGE₂tocomplete the maturation process. After 24 hrs, each DC population wasused in ELISpot assays to recruit an anti-flu M1 recall responses, asdetermined by the frequency of responding T-cells secreting IFN-γ. DCsmatured by transfection with CD40L mRNA in the presence of IFN-γ andPGE₂ invoked a more potent anti-flu response.

FIG. 10 shows that DCs matured via transfection with CD40L mRNA andcultured in the presence of IFN-γ and PGE₂ invoke efficient “primaryT-cell responses” when compared to DCs matured in the presence of the“cytokine cocktail”. DCs were transfected with 2 μg MART-APL mRNA permillion cells as antigen payload, and subsequently matured with cytokinecocktail. Alternatively, DCs were co-transfected with 2 μg MART-APL mRNAper million cells as antigen payload, and 4 μg CD40L mRNA as thematuration payload. These latter cells were immediately cultured in 1000U/ml IFN-γ and 1 μg/m1PGE₂ to complete the maturation process. After 24hrs, each DC population was used to raise T-cell responses to MART-APLpeptide sequences, generated from the transfected MART-APL mRNA payload,by co-culture of autologous naive CD8+ T-cells for 7 days in thepresence of 0.2 U/ml of IL-2. After this first round of stimulation,T-cells were harvested and established in IL-2 ELISpot assays,restimulated with the appropriately matured, antigen loaded DCs. DCsmatured by transfection with CD40L mRNA in the presence of IFN-γ andPGE₂ invoked a more potent anti-MART-APL response as determined by thefrequency of responder CD8+ Tcells secreting IL-2.

FIGS. 11A and 11B show the induction of cytotoxic T-cells by DCsexpressing MART-APL mRNA. FIG. 11A shows that maturation of DCs usingco-transfection with MART-APL mRNA as source of antigen, and CD40L mRNA,with the addition of soluble interferon-γ/PGE₂ invokes an effective CTLresponse, whereas FIG. 11B shows that DCs transfected with MART-APLmRNA, but matured with a ‘cytokine cocktail’, do not. T2-PSA: T2 cellspulsed with an HLA-A2 restricted peptide from prostate-specificantigen(PSA) as a negative control target. MART-T2: T2 cells pulsed with theHLA-A2 restricted MART epitope in its native sequence. MART-APL-T2: T2cells pulsed with the HLA-A2 restricted MART epitope as the preferred‘altered peptide ligand’.

FIG. 12 shows the migratory capacity of PME-CD40L matured DCs intranswell assays to the lymph node chemokines, CCL19 and 21. Fourindependent healthy donors were tested in parallel, with each DCpreparation being transfected with lug amplified total RCC tumor RNA,along with 4 ug CD40L RNA per million DCs. Migration assays were set up24 hrs post transfection with the mRNA payloads.

FIG. 13 shows the induction of CTL responses from a healthy donor to themelanoma-associated antigen, MART-1. DCs were prepared and loaded withMART-1 RNA and matured via the the ‘CD40L base process’ or DCs wereprepared using the PME-CD40L process. DCs and purified CD8 T-cells wereco-cultured in a 1:10 ratio, undergoing three rounds of stimulation inthe presence of IL-2. The data shows ⁵¹Cr release cytotoxic assays usingMART-1 peptide pulsed T2 target cells across a range of effector-targetratios.

FIG. 14 shows the induction of a fully autologous CTL response to DCsloaded with total amplified RCC tumor RNA, PME-CD40L matured DCs. DCsand purified CD8 T-cells were co-cultured in a 1:10 ratio, undergoingthree rounds of stimulation in the presence of IL-2. 5 days after thelast stimulation, CD8 T-cells were restimulated with DCs transfectedwith: total amplified RCC RNA, hTERT RNA, Survivin RNA, G250 RNA ornegative control DCs transfected with eGFP RNA. The data is derived fromidentifying responder T-cells by cell surface staining for theactivation marker, CD69, and simultaneously detection of intracellularIFN-γ and IL-2. Intracellular cytokine responses were subdivided toidentify IFN-γ single positive (effector T cells) from IFN-γ/IL-2 doublepositive (memory T cells).

FIGS. 15A and 15B show the effect of MART-1 RNA transfected CD40L baseprocess matured DCs pulsed with KRN7000 or vehicle on the expansion ofNKT cells (FIG. 15A) and MART-1-reactive CTLs (FIG. 15B). The dataclearly shows that KRN7000 pulsed DC can expand NKT-cells as defined byCD1d/KRN7000 tetramer staining, and that the presence of an expandedpopulation of NKT-cells can increase the concominant recruitment ofprimary CTLs to MART-1, as defined by tetramer staining withMART-1/HLA-A2 tetramers.

FIGS. 16A, 16B, and 16C show the alignment of the human (SEQ ID NO: 1)and mouse (SEQ ID NO: 9) CD40L cDNAs. FIGS. 16A, 16B and 16C represent 3consecutive pages of the alignment of SEQ ID NO: 1 and 9.

FIG. 17 shows the alignment of the human (SEQ ID NO: 2) and mouse (SEQID NO: 10) CD40L proteins.

FIG. 18 shows the level of IL-12 expression by DC transfected with mRNAtranscribed from pCR2.1 CD40L WT Delta X-E plasmid in 100 μg scale(Delta X-E1) or 1 mg scale (Delta X-E2) transcription reactions usingmMessage mMachine T7 Ultra kit (Ambion). Reference RNA was transcribedfrom plasmid pCR2.1 CD40L WT. The transcribed CD40L RNAs were modifiedby addition of polyA tail using polyA plus kit (Epicentre). RNAs weretransfected into DCs. Approximately 20 hrs post transfection the amountof IL-12 was measured in the supernatant of the matured DCs using Elisa.Negative control: IL-12 expression measured in the supernatant of DCselectroporated without any CD40L RNA.

FIG. 19 shows the level of IL-12 in supernatants of DC culturetransfected with various RNA constructs. In order to assess the impactof the various 5′UTR sequences on CD40L protein expression and theinduction of IL-12 cytokine, three RNAs were generated from the plasmidspCR2.1 CD40L WT, pCR2.1 CD40L ΔE, and pCR2.1 CD40L+5UTR using themMessage mMachine T7 Ultra transcription kit. The transcribed RNAs werepolyadenylated and purified using an RNeasy kit (Qiagen). The purifiedRNAs were transfected into mDCs. IL-12 cytokine induction in the DCculture was measured by ELISA in collected supernatants.

FIG. 20 shows SDS-PAGE resolution of in vitro translated[³⁵S]-methionine labeled CD40L protein derived from RNAs containingvarious 5′UTRs.

FIG. 21 shows SDS-PAGE resolution of [³⁵S]-methionine labeled CD40Lproteins in vitro translated from mRNAs containing normal and mutatedstart codons.

FIG. 22 shows dendritic cells transfected with various CD40L RNAs andstained with an anti-CD154 (CD40L) antibody. Left Panel: Percentage ofCD40L positive cells after 4 hours. Right panel: Mean Fluorescentintensity of CD40L staining. CD40L WT, the original RNA construct,serves as a positive control. GFP RNA transfected cells serve as anegative control.

FIG. 23 shows the expression profile of IL-10 and IL-12 in DCtransfected with various CD40L RNAs.

FIG. 24 shows the isoforms of the in vitro translation products derivedfrom various CD40L mRNAs. The table in this figure shows amount of IL-12cytokine expressed by dendritic cells transfected with these CD40LmRNAs.

FIG. 25 shows secretion of IL-10 and IL-12 by dendritic cellstransfected with the indicated modified CD40L RNAs.

FIG. 26 shows SDS-PAGE resolution of the translation products of theCD40L polypeptides produced from the indicated CD40L RNAs.

FIG. 27 shows the secretion levels of IL-10 and IL-12 by Dendritic cellstransfected with the indicated modified CD40L RNAs.

FIG. 28 shows the increased percentage of Mart-1 reactive CTL on day 10in co-cultures with DC generated with the PME-CD40L process compared toother methods of generating DC such as DC electroporated with CD40L RNAand Mart-1 RNA and cultured for 4 hours with IFN-γ and PGE₂ (CD40L) orDC matured with cytokines (TFNα, IFN-γ and PGE₂) overnight thenelectroported with Mart-1 RNA and cultured for 4 hours (TIP) or immatureDC electroported with MART-1 RNA and co-cultured with cytokine cocktail(IL-6, IL-1β, TFNα, IFNγ, PGE₂) for 4 hours (Cytokines).

FIG. 29 shows the time course of CD28 receptor expression in MART-1 CTLco-cultured with DCs prepared by the PME-CD40L process, TIP process,CD40L base process or the cytokine cocktail process.

FIG. 30 shows that PME-CD40L generated DC in contrast to other methodsof generating mature DC are capable of priming MART-1 specific CTL thatretain the capacity to produce both IL-2 and IFN-γ.

FIG. 31 shows the mean fluorescence intensity (MFI) of IFN-γ positiveCTL as a measure of the overall level of cytokine being produced byMart-1 CTL.

MODES FOR CARRYING OUT THE INVENTION

Throughout this disclosure, various publications, patents and publishedpatent specifications are referenced by an identifying citation. Thedisclosures of these publications, patents and published patentspecifications are hereby specifically incorporated by reference intothe present disclosure to more fully describe the state of the art towhich this invention pertains.

The practice of the present invention employs, unless otherwiseindicated, conventional techniques of molecular biology (includingrecombinant techniques), microbiology, cell biology, biochemistry andimmunology, which are within the skill of the art. Such techniques areexplained fully in the literature. These methods are described in thefollowing publications. See, e.g., Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2^(nd) edition (1989); CURRENT PROTOCOLS IN MOLECULARBIOLOGY (Ausubel et al. eds. (1987)); the series METHODS IN ENZYMOLOGY(Academic Press, Inc.); PCR: A PRACTICAL APPROACH (M. MacPherson et al.IRL Press at Oxford University Press (1991)); PCR 2: A PRACTICALAPPROACH (MacPherson, Hames and Taylor eds. (1995)); ANTIBODIES, ALABORATORY MANUAL (Harlow and Lane eds. (1988)); USING ANTIBODIES, ALABORATORY MANUAL (Harlow and Lane eds. (1999)); and ANIMAL CELL CULTURE(Freshney ed. (1987)).

Definitions

As used in the specification and claims, the singular form “a,” “an” and“the” include plural references unless the context clearly dictatesotherwise. For example, the term “a cell” includes a plurality of cells,including mixtures thereof.

As used herein, the term “comprising” is intended to mean that thecompositions and methods include the recited elements, but not excludingothers. “Consisting essentially of” when used to define compositions andmethods, shall mean excluding other elements of any essentialsignificance to the combination. Thus, a composition consistingessentially of the elements as defined herein would not exclude tracecontaminants from the isolation and purification method andpharmaceutically acceptable carriers, such as phosphate buffered saline,preservatives, and the like. Polypeptides or protein that “consistessentially of” a given amino acid sequence are defined herein tocontain no more than three, preferably no more than two, and mostpreferably no more than one additional amino acids at the amino and/orcarboxy terminus of the protein or polypeptide. Nucleic acids orpolynucleotides that “consist essentially of” a given nucleic acidsequence are defined herein to contain no more than ten, preferably nomore than six, more preferably no more than three, and most preferablyno more than one additional nucleotide at the 5′ or 3′ terminus of thenucleic acid sequence. “Consisting of” shall mean excluding more thantrace elements of other ingredients and substantial method steps foradministering the compositions of this invention. Embodiments defined byeach of these transition terms are within the scope of this invention.

All numerical designations, e.g., pH, temperature, time, concentration,and molecular weight, including ranges, are approximations which arevaried (+) or (−) by increments of 0.1. It is to be understood, althoughnot always explicitly stated, that the reagents described herein aremerely exemplary and that equivalents of such are known in the art.

The term “antigen” is well understood in the art and includes substanceswhich are immunogenic, i.e., immunogen. It will be appreciated that theuse of any antigen is envisioned for use in the present invention andthus includes, but is not limited to a self-antigen (whether normal ordisease-related), an infectious antigen (e.g., a microbial antigen,viral antigen, etc.), or some other foreign antigen (e.g., a foodcomponent, pollen, etc.). The term “antigen” or alternatively,“immunogen” applies to collections of more than one immunogen, so thatimmune responses to multiple immunogens may be modulated simultaneously.Moreover, the term includes any of a variety of different formulationsof immunogen or antigen.

A “native” or “natural” or “wild-type” antigen is a polypeptide, proteinor a fragment which contains an epitope, which has been isolated from anatural biological source, and which can specifically bind to an antigenreceptor, when presented as an MHC/peptide complex, in particular a Tcell antigen receptor (TCR), in a subject.

The term “tumor associated antigen” or “TAA” refers to an antigen thatis associated with a tumor. Examples of well known TAAs include gp100,MART and MAGE.

The terms “major histocompatibility complex” or “MHC” refers to acomplex of genes encoding cell-surface molecules that are required forantigen presentation to T cells and for rapid graft rejection. Inhumans, the MHC is also known as the “human leukocyte antigen” or “HLA”complex. The proteins encoded by the MHC are known as “MHC molecules”and are classified into Class I and Class II MHC molecules. Class I MHCmolecules include membrane heterodimeric proteins made up of an α chainencoded in the MHC noncovalently linked with the β₂-microglobulin. ClassI MHC molecules are expressed by nearly all nucleated cells and havebeen shown to function in antigen presentation to CD8¹ T cells. Class Imolecules include HLA-A, B, and C in humans. Class II MHC molecules alsoinclude membrane heterodimeric proteins consisting of noncovalentlyassociated α and β chains. Class II MHC molecules are known to functionin CD4⁺ T cells and, in humans, include HLA-DP, -DQ, and -DR.

The term “antigen presenting cells (APCs)” refers to a class of cellscapable of presenting one or more antigens in the form of peptide-MHCcomplex recognizable by specific effector cells of the immune system,and thereby inducing an effective cellular immune response against theantigen or antigens being presented. APCs can be intact whole cells suchas macrophages, B-cells, endothelial cells, activated T-cells, anddendritic cells; or other molecules, naturally occurring or synthetic,such as purified MHC Class I molecules complexed to β2-microglobulin.While many types of cells may be capable of presenting antigens on theircell surface for T-cell recognition, only dendritic cells have thecapacity to present antigens in an efficient amount to activate naiveT-cells for cytotoxic T-lymphocyte (CTL) responses.

The term “dendritic cells (DCs)” refers to a diverse population ofmorphologically similar cell types found in a variety of lymphoid andnon-lymphoid tissues, Steinman (1991) Ann. Rev. Immunol. 9:271-296.Dendritic cells constitute the most potent and preferred APCs in theorganism. While the dendritic cells can be differentiated frommonocytes, they possess distinct phenotypes. For example, a particulardifferentiating marker, CD14 antigen, is not found in dendritic cellsbut is possessed by monocytes. Also, mature dendritic cells are notphagocytic, whereas the monocytes are strongly phagocytosing cells. Ithas been shown that mature DCs can provide all the signals necessary forT cell activation and proliferation.

The term “immune effector cells” refers to cells capable of binding anantigen and which mediate an immune response. These cells include, butare not limited to, T cells, B cells, monocytes, macrophages, NK cellsand cytotoxic T lymphocytes (CTLs), for example CTL lines, CTL clones,and CTLs from tumor, inflammatory, or other infiltrates.

A “naïve” immune effector cell is an immune effector cell that has neverbeen exposed to an antigen capable of activating that cell. Activationof naive immune effector cells requires both recognition of thepeptide:MHC complex and the simultaneous delivery of a costimulatorysignal by a professional APC in order to proliferate and differentiateinto antigen-specific armed effector T cells.

“Immune response” broadly refers to the antigen-specific responses oflymphocytes to foreign substances. Any substance that can elicit animmune response is said to be “immunogenic” and is referred to as an“immunogen”. All immunogens are antigens, however, not all antigens areimmunogenic. An immune response of this invention can be humoral (viaantibody activity) or cell-mediated (via T cell activation).

As used herein, the term “educated, antigen-specific immune effectorcell”, is an immune effector cell as defined above, which has previouslyencountered an antigen. In contrast to its naïve counterpart, activationof an educated, antigen specific immune effector cell does not require acostimulatory signal. Recognition of the peptide: MHC complex issufficient.

“Activated”, when used in reference to a T cell, implies that the cellis no longer in G_(o) phase, and begins to produce one or more ofcytotoxins, cytokines and other related membrane-associated proteinscharacteristic of the cell type (e.g., CD8⁺or CD4⁺), and is capable ofrecognizing and binding any target cell that displays the particularpeptide/MHC complex on its surface, and releasing its effectormolecules.

As used herein, the term “inducing an immune response in a subject” is aterm understood in the art and refers to an increase of at least about2-fold, or alternatively at least about 5-fold, or alternatively atleast about 10-fold, or alternatively at least about 100-fold, oralternatively at least about 500-fold, or alternatively at least about1000-fold or more in an immune response to an antigen (or epitope) whichcan be detected or measured, after introducing the antigen (or epitope)into the subject, relative to the immune response (if any) beforeintroduction of the antigen (or epitope) into the subject. An immuneresponse to an antigen (or epitope), includes but is not limited to,production of an antigen-specific (or epitope-specific) antibody, andproduction of an immune cell expressing on its surface a molecule whichspecifically binds to an antigen (or epitope). Methods of determiningwhether an immune response to a given antigen (or epitope) has beeninduced are well known in the art. For example, antigen-specificantibody can be detected using any of a variety of immunoassays known inthe art, including, but not limited to, ELISA, wherein, for example,binding of an antibody in a sample to an immobilized antigen (orepitope) is detected with a detectably-labeled second antibody (e.g.,enzyme-labeled mouse anti-human Ig antibody).

“Co-stimulatory molecules” are involved in the interaction betweenreceptor-ligand pairs expressed on the surface of antigen presentingcells and T cells. Research accumulated over the past several years hasdemonstrated convincingly that resting T cells require at least twosignals for induction of cytokine gene expression and proliferation(Schwartz, R. H. (1990) Science 248: 1349-1356 and Jenkins, M. K. (1992)Immunol. Today 13:69-73). One signal, the one that confers specificity,can be produced by interaction of the TCR/CD3 complex with anappropriate MHC/peptide complex. The second signal is not antigenspecific and is termed the “co-stimulatory” signal. This signal wasoriginally defined as an activity provided by bone-marrow-derivedaccessory cells such as macrophages and dendritic cells, the so called“professional” APCs. Several molecules have been shown to enhanceco-stimulatory activity. These are heat stable antigen (HSA) (Liu, Y. etal. (1992) 3. Exp. Med. 175:437-445), chondroitin sulfate-modified MHCinvariant chain (li-CS) (Naujokas, M. F. et al. (1993) Cell 74:257-268),intracellular adhesion molecule 1 (ICAM-1) (Van Seventer, G. A. (1990)].Immunol. 144:4579-4586), B7-1, and B7-2/B70 (Schwartz, R. H. (1992) Cell71:1065-1068). These molecules each appear to assist co-stimulation byinteracting with their cognate ligands on the T cells. Co-stimulatorymolecules mediate co-stimulatory signal(s), which are necessary, undernormal physiological conditions, to achieve full activation of naïve Tcells. One exemplary receptor-ligand pair is the B7 family ofco-stimulatory molecule on the surface of APC5 and its counterreceptorCD28 or CTLA-4 on T cells (Freeman, et al. (1993) Science 262:909-911;Young, et al. (1992)]. Clin. Invest. 90:229 and Nabavi, et al. (1992)Nature 360:266-268). Other important co-stimulatory molecules are CD40,and CD54. The term “costimulatory molecule” encompasses any singlemolecule or combination of molecules which, when acting together with aMHC/peptide complex bound by a TCR on the surface of a T cell, providesa co-stimulatory effect which achieves activation of the I cell thatbinds the peptide. The term thus encompasses B7, or other co-stimulatorymolecule(s) on an antigen-presenting matrix such as an APC, fragmentsthereof (alone, complexed with another molecule(s), or as part of afusion protein) which, together with MHC complex, binds to a cognateligand and results in activation of the T cell when the TCR on thesurface of the T cell specifically binds the peptide. It is intended,although not always explicitly stated, that molecules having similarbiological activity as wild-type or purified co-stimulatory molecules(e.g., recombinantly produced or muteins thereof) are intended to beused within the spirit and scope of the invention.

As used herein, the term “cytokine” refers to any one of the numerousfactors that exert a variety of effects on cells, for example, inducinggrowth or proliferation. Non-limiting examples of cytokines which may beused alone or in combination in the practice of the present inventioninclude, interleukin-2 (IL-2), stem cell factor (SCF), interleukin-3(IL-3), interleukin-6 (IL-6), interleukin-12 (IL-12), G-CSF, granulocytemacrophage-colony stimulating factor (GM-CSF), interleukin-1 alpha(IL-1α), interleukin-1L (IL-11), MIP-11, leukemia inhibitory factor(LIF), c-kit ligand, thrombopoietin (TPO) and flt3 ligand. Oneembodiment of the present invention includes culture conditions in whichan effective amount of IL-1β and/or IL-6 is excluded from the medium.Cytokines are commercially available from several vendors such as, forexample, Genzyme (Framingham, Mass.), Genentech (South San Francisco,Calif.), Amgen (Thousand Oaks, Calif.), R&D Systems (Minneapolis, Minn.)and Immunex (Seattle, Wash.). It is intended, although not alwaysexplicitly stated, that molecules having similar biological activity aswild-type or purified cytokines (e.g., recombinantly produced or muteinsthereof) are intended to be used within the spirit and scope of theinvention.

The terms “polynucleotide”, “nucleic acid” and “nucleic acid molecule”are used interchangeably to refer to polymeric forms of nucleotides ofany length. The polynucleotides may contain deoxyribonucleotides,ribonucleotides, and/or their analogs. Nucleotides may have anythree-dimensional structure, and may perform any function, known orunknown. The term “polynucleotide” includes, for example,single-stranded, double-stranded and triple helical molecules, a gene orgene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA,recombinant polynucleotides, branched polynucleotides, plasmids,vectors, isolated DNA of any sequence, isolated RNA of any sequence,nucleic acid probes, and primers. In addition to a native nucleic acidmolecule, a nucleic acid molecule of the present invention may alsocomprise modified nucleic acid molecules. As used herein, mRNA refers toan RNA that can be translated in a dendritic cell. Such mRNAs typicallyare capped and have a ribosome binding site (Kozak sequence) and atranslational initiation codon.

The term “peptide” is used in its broadest sense to refer to a compoundof two or more subunit amino acids, amino acid analogs, orpeptidomimetics. The subunits may be linked by peptide bonds. In anotherembodiment, the subunit may be linked by other bonds, e.g., ester,ether, etc. As used herein the term “amino acid” refers to eithernatural and/or unnatural or synthetic amino acids, including glycine andboth the D and L optical isomers, amino acid analogs andpeptidomimetics. A peptide of three or more amino acids is commonlycalled an oligopeptide if the peptide chain is short. If the peptidechain is long, the peptide is commonly called a polypeptide or aprotein.

The term “genetically modified” means containing and/or expressing aforeign gene or nucleic acid sequence which in turn, modifies thegenotype or phenotype of the cell or its progeny. In other words, itrefers to any addition, deletion or disruption to a cell's endogenousnucleotides.

As used herein, “expression” refers to the processes by whichpolynucleotides are transcribed into mRNA and mRNA is translated intopeptides, polypeptides, or proteins. If the polynucleotide is derivedfrom genomic DNA of an appropriate eukaryotic host expression mayinclude splicing of the mRNA. Regulatory elements required forexpression include promoter sequences to bind RNA polymerase andtranscription initiation sequences for ribosome binding. For example, abacterial expression vector includes a promoter such as the lac promoterand for transcription initiation the Shine-Dalgarno sequence and thestart codon AUG (Sambrook et al. (1989) supra). Similarly, a eukaryoticexpression vector includes a heterologous or homologous promoter for RNApolymerase II, a downstream polyadenylation signal, the start codon AUG,and a termination codon for detachment of the ribosome. Such vectors canbe obtained commercially or assembled by the sequences described inmethods known in the art, for example, the methods herein below forconstructing vectors in general.

“Under transcriptional control” is a term understood in the art andindicates that transcription of a polynucleotide sequence, usually a DNAsequence, depends on its being operatively linked to an element whichcontributes to the initiation of, or promotes, transcription.“Operatively linked” refers to a juxtaposition wherein the elements arein an arrangement allowing them to function.

A “gene delivery vehicle” is defined as any molecule that can carryinserted polynucleotides into a host cell. Examples of gene deliveryvehicles are liposomes, biocompatible polymers, including naturalpolymers and synthetic polymers; lipoproteins; polypeptides;polysaccharides; lipopolysaccharides; artificial viral envelopes; metalparticles; and bacteria, or viruses, such as baculovirus, adenovirus andretrovirus, bacteriophage, cosmid, plasmid, fungal vectors and otherrecombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

“Gene delivery,” “gene transfer,” “transfection” and the like as usedherein, are terms referring to the introduction of an exogenouspolynucleotide into a host cell, irrespective of the method used for theintroduction. Transfection refers to delivery of any nucleic acid to theinterior of a cell. Gene delivery refers to the delivery of a nucleicacid that may be integrated into the host cell's genome, or that mayreplicate independently of the host cell genome. Gene delivery or genetransfer does not refer to introduction of an mRNA into a cell.Transfection methods include a variety of techniques such aselectroporation, protein-based, lipid-based and cationic ion basednucleic acid delivery complexes, viral vectors, “gene gun” delivery andvarious other techniques known to those of skill in the art. Theintroduced polynucleotide can be stably maintained in the host cell ormay be transiently expressed. In preferred embodiments, an mRNA isintroduced into a DC and is transiently expressed. Stable maintenancetypically requires that the introduced polynucleotide either contains anorigin of replication compatible with the host cell or integrates into areplicon of the host cell such as an extrachromosomal replicon (e.g., aplasmid) or a nuclear or mitochondrial chromosome. A number of vectorsare capable of mediating transfer of genes to mammalian cells, as isknown in the art and described herein.

A “viral vector” is defined as a recombinantly produced virus or viralparticle that comprises a polynucleotide to be delivered into a hostcell, either in vivo, ex vivo or in vitro. Examples of viral vectorsinclude retroviral vectors, adenovirus vectors, adeno-associated virusvectors, alphavirus vectors and the like. Alphavirus vectors, such asSemliki Forest virus-based vectors and Sindbis virus-based vectors, havealso been developed for use in gene therapy and immunotherapy. See,Schlesinger and Dubensky (1999) Curr. Opin. Biotechnol. 5:434-439 andZaks et al. (1999) Nat. Med. 7:823-827. In aspects where gene transferis mediated by a retroviral vector, a vector construct refers to thepolynucleotide comprising the retroviral genome or part thereof, and atherapeutic gene. As used herein, “retroviral mediated gene transfer” or“retroviral transduction” carries the same meaning and refers to theprocess by which a gene or nucleic acid sequences are stably transferredinto the host cell by virtue of the virus entering the cell andintegrating its genome into the host cell genome. The virus can enterthe host cell via its normal mechanism of infection or be modified suchthat it binds to a different host cell surface receptor or ligand toenter the cell. As used herein, “retroviral vector” refers to a viralparticle capable of introducing exogenous nucleic acid into a cellthrough a viral or viral-like entry mechanism.

Retroviruses carry their genetic information in the form of RNA;however, once the virus infects a cell, the RNA is reverse-transcribedinto the DNA form which integrates into the genomic DNA of the infectedcell. The integrated DNA form is called a provirus.

In aspects where gene transfer is mediated by a DNA viral vector, suchas an adenovirus (Ad), pseudo adenoviral or adeno-associated virus (MV),vector construct refers to the polynucleotide comprising the viralgenome or part thereof, and a transgene. Adenoviruses (Ads) are arelatively well characterized, homogenous group of viruses, includingover 50 serotypes. (See, e.g., WO 95/27071). Ads are easy to grow and donot require integration into the host cell genome. RecombinantAd-derived vectors, particularly those that reduce the potential forrecombination and generation of wild-type virus, have also beenconstructed. (See, WO 95/00655 and WO 95/11984). Wild-type MV has highinfectivity and specificity integrating into the host cell's genome.(See, Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996).

Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression.

Gene delivery vehicles also include several non-viral vectors, includingDNA/liposome complexes, and targeted viral protein-DNA complexes.Liposomes that also comprise a targeting antibody or fragment thereofcan be used in the methods of this invention. To enhance delivery to acell, nucleic acids or proteins of this invention can be conjugated toantibodies or binding fragments thereof which bind cell surfaceantigens, e.g., TCR, CD3 or CD4.

“Hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson-Crick base pairing, Hoogstein binding, or inany other sequence-specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming amulti-stranded complex, a single self-hybridizing strand, or anycombination of these. A hybridization reaction may constitute a step ina more extensive process, such as the initiation of a PCR reaction, orthe enzymatic cleavage of a polynucleotide by a ribozyme.

Stringent hybridization conditions are as follows: Prehybridization offilters containing a nucleic acid of interest is carried out for 8 hrsto overnight at 65° C. in buffer composed of 6×SSC, 50 mM Tris-HCl (pH7.5), 1 mM EDTA, 0.02% Ficoll, 0.02% BSA, and 500 μg/m1 denatured salmonsperm DNA. Filters are hybridized for 48 hrs at 65° C., the preferredhybridization temperature, in prehybridization mixture containing 100μg/ml denatured salmon sperm DNA and 5×20×10⁶ cpm of ³²P-labeled probe.Subsequently, filter washes are performed at 37° C. for 1 h in asolution containing 2×SSC, 0.01% Ficoll, and 0.01% BSA, followed by awash in 0.1×SSC at 50° C. for 45 min. Following the wash steps, thehybridized probes are detectable by autoradiography. Such methods arewell known in the art and cited in Sambrook et al., 1989; and Ausubel etal., 1989.

A polynucleotide or polynucleotide region (or a polypeptide orpolypeptide region) has a certain percentage (for example, 80%, 85%,90%, or 95%) of “sequence identity” to another sequence means that, whenaligned, that percentage of bases (or amino acids) are the same incomparing the two sequences. This alignment and the percent homology orsequence identity is be determined using the well known BLAST alignmentprogram and the default parameters. Alternative programs are BLASTN andBLASTP, using the following default parameters: Genetic code=standard;filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62;Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant,GenBank+EMBL+DDBJ+PDB+GenBank CDStranslations+SwissProtein+SPupdate+PIR. Details of these programs can befound at the following world wide web address:ncbi.nlm.nih.gov/cgi-bin/BLAST.

The term “isolated” means separated from constituents, cellular andotherwise, in which the polynucleotide, peptide, polypeptide, protein,antibody, or fragments thereof, are normally associated with in nature.For example, with respect to a polynucleotide, an isolatedpolynucleotide is one that is separated from the 5′ and 3′ sequenceswith which it is normally associated in the chromosome. As is apparentto those of skill in the art, a non-naturally occurring polynucleotide,peptide, polypeptide, protein, antibody, or fragment(s) thereof, doesnot require “isolation” to distinguish it from its naturally occurringcounterpart. In addition, a “concentrated”, “separated” or “diluted”polynucleotide, peptide, polypeptide, protein, antibody, or fragment(s)thereof, is distinguishable from its naturally occurring counterpart inthat the concentration or number of molecules per volume is greater than“concentrated” or less than “separated” than that of its naturallyoccurring counterpart. A polynucleotide, peptide, polypeptide, protein,antibody, or fragment(s) thereof, which differs from the naturallyoccurring counterpart in its primary sequence or for example, by itsglycosylation pattern, need not be present in its isolated form since itis distinguishable from its naturally occurring counterpart by itsprimary sequence, or alternatively, by another characteristic such asits glycosylation pattern. Although not explicitly stated for each ofthe inventions disclosed herein, it is to be understood that all of theabove embodiments for each of the compositions disclosed below and underthe appropriate conditions, are provided by this invention. Thus, anon-naturally occurring polynucleotide is provided as a separateembodiment from the isolated naturally occurring polynucleotide. Aprotein produced in a bacterial cell is provided as a separateembodiment from the naturally occurring protein isolated from aeukaryotic cell in which it is produced in nature. A mammalian cell,such as dendritic cell is isolated if it is removed from the anatomicalsite from which it is found in an organism.

“Host cell,” “target cell” or “recipient cell” are intended to includeany individual cell or cell culture which can be or have been recipientsfor vectors or the incorporation of exogenous nucleic acid molecules,polynucleotides and/or proteins. It also is intended to include progenyof a single cell, and the progeny may not necessarily be completelyidentical (in morphology or in genomic or total DNA complement) to theoriginal parent cell due to natural, accidental, or deliberate mutation.The cells may be prokaryotic or eukaryotic, and include but are notlimited to bacterial cells, yeast cells, animal cells, and mammaliancells, e.g., murine, rat, simian or human.

A “subject” is a vertebrate, preferably a mammal, more preferably ahuman. Mammals include, but are not limited to, murines, simians,humans, farm animals, sport animals, and pets.

A “control” is an alternative subject or sample used in an experimentfor comparison purpose. A control can be “positive” or “negative”. Forexample, where the purpose of the experiment is to determine acorrelation of an immune response with a particular culture condition,it is generally preferable to use a positive control and a negativecontrol.

By “cancer” is meant the abnormal presence of cells which exhibitrelatively autonomous growth, so that a cancer cell exhibits an aberrantgrowth phenotype characterized by a significant loss of cellproliferation control. Cancerous cells can be benign or malignant. Invarious embodiments, the cancer affects cells of the bladder, blood,brain, breast, colon, digestive tract, lung, ovaries, pancreas, prostategland, or skin. The definition of a cancer cell, as used herein,includes not only a primary cancer cell, but also any cell derived froma cancer cell ancestor. This includes metastasized cancer cells, and invitro cultures and cell lines derived from cancer cells. Cancerincludes, but is not limited to, solid tumors, liquid tumors,hematologic malignancies, renal cell cancer, melanoma, breast cancer,prostate cancer, testicular cancer, bladder cancer, ovarian cancer,cervical cancer, stomach cancer, esophageal cancer, pancreatic cancer,lung cancer, neuroblastoma, glioblastoma, retinoblastoma, leukemias,myelomas, lymphomas, hepatoma, adenomas, sarcomas, carcinomas,blastomas, etc. When referring to a type of cancer that normallymanifests as a solid tumor, a “clinically detectable” tumor is one thatis detectable on the basis of tumor mass; e.g., by such procedures asCAT scan, magnetic resonance imaging (MRI), X-ray, ultrasound orpalpation. Biochemical or immunologic findings alone may be insufficientto meet this definition.

The term “culturing” refers to the in vitro maintenance,differentiation, and/or propagation of cells or in suitable media. By“enriched” is meant a composition comprising cells present in a greaterpercentage of total cells than is found in the tissues where they arepresent in an organism. For example, the enriched cultures andpreparations of CD83⁺ CCR⁻ DCs and CD83⁺ CCR7⁺ DCs made by the methodsof the invention are present in a higher percentage of total cells ascompared to their percentage in the tissues where they are present in anorganism (e.g., blood, skin, lymph nodes, etc.).

A “composition” is intended to mean a combination of active agent andanother compound or composition, inert (for example, a detectable agentor label) or active, such as an adjuvant.

A “pharmaceutical composition” is intended to include the combination ofan active agent with a carrier, inert or active, making the compositionsuitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

As used herein, the term “pharmaceutically acceptable carrier”encompasses any of the standard pharmaceutical carriers, such as aphosphate buffered saline solution, water, and emulsions, such as anoil/water or water/oil emulsion, and various types of wetting agents.The compositions also can include stabilizers and preservatives. Forexamples of carriers, stabilizers and adjuvants, see Martin REMINGTON′SPHARM. SCI., 18th Ed. (Mack Publ. Co., Easton (1990)).

An “effective amount” is an amount sufficient to effect beneficial ordesired results, such as enhanced immune response, treatment, preventionor amelioration of a medical condition (disease, infection, etc). Aneffective amount can be administered in one or more administrations,applications or dosages. Suitable dosages will vary depending on bodyweight, age, health, disease or condition to be treated and route ofadministration.

As used herein, “signaling” means contacting an immature or maturedendritic cell with an IFN-γ receptor agonist, a TNF-α receptor agonist,a CD40L polypeptide or other CD40 agonist. In one embodiment, suchagonists are provided externally, (e.g., in the cell culture medium). Inanother embodiment, the polypeptide agonist is provided via transfectionof an immature or mature dendritic cell with a nucleic acid encoding thepolypeptide. Alternatively, a nucleic acid aptamer agonist could beprovided in the medium or by transfection. In cases where thepolypeptide(s) is provided by transfecting a dendritic cell with anucleic acid encoding the polypeptide, signaling is effected upontranslation of an mRNA encoding the polypeptide, rather than upontransfection with the nucleic acid. In one aspect, this inventionprovides methods for preparing enriched populations of mature dendriticcells (DCs) that induce potent immunostimulatory responses in vivoand/or in vitro. As used herein, the term “mature dendritic cells” meansdendritic cells that demonstrate elevated cell surface expression ofco-stimulator molecule CD83, compared to immature DCs (iDCs). Mature DCsof the invention include both CD83¹ CCR7 ⁻ DCs and CD83⁺ CCR7⁺ DCs. Thesecond signal, a CD40 agonist, can be given to either immature CD83⁻CCR7 ⁻ DCs, or to CD83⁺ CCR7 ⁻ mature DCs.

The literature (Schaft 2005, Bonehill 2004) suggests that postmaturation electroporation of DCs with antigen-encoding RNA resulted inDCs with greater potency to invoke immune responses. Therefore, methodswere developed to alter the ‘CD40L base process’ (sequential IFN-γsignaling and CD40L signaling of CD83⁻ iDCs), by altering the timing ofthe CD40L signaling to CD83⁺ CCR7 ⁻ mature DCs (post phenotypicmaturation). In this embodiment, DCs were first phenotypically maturedby adding ‘inflammatory mediators’, IFN-γ and TNF-α, and optionallyPGE₂, to the culture medium, and then electroporating with CD40L mRNA,and optionally antigen-encoding mRNA approximately 12-30 hours(preferably about 18 hrs) later. This novel process was named‘PME-CD40L’, for Post Maturation Electroporation with CD40L to produceCD83¹ CCR7¹ mature DCs. Cells harvested 4 hrs post electroporation andformulated as a vaccine were shown to mediate maximum immunopotency inin vitro assays (see examples). Dendritic cells made by the PME-CD40Lprocess are phenotypically different than prior art dendritic cells. Forexample, PME-CD40L process of generating DC is capable of supportinglong term antigen specific CTL effector function and inducing apreferred phenotype of effector memory CTL that retains the capacity toexpand, produce cytokines and kill target cells all critical eventsmediating robust ling-term CTL effector function. Thus, in oneembodiment, the invention provides a dendritic cell which preferentiallyinduces a population of CD28⁺ CD45RA⁻ memory/effector T cells from apopulation of antigen-specific T cells. The antigen specific T cells canbe naïve T cells or antigen experienced T cells. Effector/memory T cellsproduce IFNγ, IL-2, and can kill target cells. Effector T cells produceIFNγ and can kill target cells, but do not produce IL-2. Memory T cellsproduce IFNγ and IL-2, but do not kill target cells.

As yet a further enhancement, DCs can be pulsed with an activationligand for NKT-cells, namely α-galactosylceramide, so as to recruit thispopulation of effector cells to the immune response. NKT-cells displayfacets of both T-helper and T-cytotoxic cells: NKT-cells can secreteIFN-γ, display CD40L, and can secrete granzyme B, the latter to induceapoptosis in target cells. Thus, NKT-cell recruitment can lead toenhanced DC function by virtue of additional NKT-cell CD40L/DC-CD40interactions, or amplify cell mediated immune responses by secretinghelper cytokines, and/or contributing to a direct lytic effect on targetcells.

After sequential signaling with the first signal (an IFN-γ receptoragonist and/or a TNF-α receptor agonist) to iDCs, and the second signal(a CD40 agonist) to either CD83⁻ CCR7 ⁻ iDCs, or to CD83⁺ CCR7 ⁻ matureDCs, the resulting DCs demonstrate (i) elevated cell surface expressionof co-stimulator molecules CD80, CD83, and CD86, ii) are CCR7⁺, and iii)secrete IL-12 p70 polypeptide or protein, and/or secrete significantlyreduced levels (0 to 500 pg/per million DCs) of IL-10. In preferredembodiments, the mature CD83⁺ CCR7¹ DCs of the invention produce atleast 1000 pg IL-12/10⁶ DCs, preferably at least 2000, 3000, 4000, 5000,or 6000 pg IL-12/10⁶ DCs, more preferably at least 7000, 8000, 9000 or10,000 pg IL-12/10⁶ DCs, and most preferably at least 12,000, 15,000,17,000 or 20,000 pg IL-12/10⁶ DCs. IL-10 and IL-12 levels can bedetermined by ELISA of culture supernatants collected at up to 36 hrspost induction of DC maturation from immature DCs. Wierda et al. (2000)Blood 96:2917. Ajdary et al. (2000) Infection and Immunity 68:1760.

Immature DCs can be isolated or prepared from a suitable tissue sourcecontaining DC precursor cells and differentiated in vitro to produceimmature DC. For example, a suitable tissue source can be one or more ofbone marrow cells, peripheral blood progenitor cells (PBPCs), peripheralblood stem cells (PBSCs), and cord blood cells. Preferably, the tissuesource is a peripheral blood mononuclear cell (PBMC). The tissue sourcecan be fresh or frozen. In another aspect, the cells or tissue sourceare pre-treated with an effective amount of a growth factor thatpromotes growth and differentiation of non-stem or progenitor cells,which are then more easily separated from the cells of interest. Thesemethods are known in the art and described briefly in Romani, et al.(1994) Exp. Med. 180:83 and Caux, C. et al. (1996) Exp. Med. 184:695. Inone aspect, the immature DCS are isolated from peripheral bloodmononuclear cells (PBMCs). In a preferred embodiment, the PBMCs aretreated with an effective amount of granulocyte macrophage colonystimulating factor (GM-CSF) in the presence or absence of interleukin 4(IL-4) and/or IL-13, so that the PBMCs differentiate into immature DCs.Most preferably, PBMCs are cultured in the presence of GM-CSF and IL-4for about 4-7 days, preferably about 5-6 days, to produce immature DCs.In preferred embodiments, the first signal is given at day 4, 5, 6, or7, and most preferably at day 5 or 6. In addition, GM-CSF as well asIL-4 and/or IL-13 may be present in the medium at the time of the firstand/or second signaling.

To increase the number of dendritic precursor cells in animals,including humans, one can pre-treat subjects with substances whichstimulate hematopoiesis. Such substances include, but are not limited toG-CSF, and GM-CSF. The amount of hematopoietic factor to be administeredmay be determined by one skilled in the art by monitoring the celldifferential of individuals to whom the factor is being administered.Typically, dosages of factors such as G-CSF and GM-CSF will be similarto the dosage used to treat individuals recovering from treatment withcytotoxic agents. As an example, GM-CSF or G-CSF can be administered for4 to 7 days at standard doses prior to removal of source tissue toincrease the proportion of dendritic cell precursors. U.S. Pat. No.6,475,483 teaches that dosages of G-CSF of 300 micrograms daily for 5 to13 days and dosages of GM-CSF of 400 micrograms daily for 4 to 19 daysresult in significant yields of dendritic cells.

The methods of the invention produce an enriched population of matureCD83⁺ CCR7⁺ dendritic cells that are potent immunostimulatory agents.Specifically, the invention provides a method for preparing maturedendritic cells (DCs), comprising the sequential steps of: (a) signalingisolated immature dendritic cells (iDCs) with a first signal comprisingan interferon gamma receptor (IFN-γR) agonist, and optionally a TNF-αRagonist, to produce IFN-γR agonist signaled dendritic cells; and (b)signaling said IFN-γR agonist signaled dendritic cells with a secondtransient signal comprising an effective amount of a CD40 agonist toproduce CCR7⁺ mature dendritic cells. The invention further providesCD83⁺ CCR7 ⁻ mature DCs and CD83⁺ CCR7⁺ mature DCs. In preferredembodiments, the CD83⁺ CCR7⁺ mature DCs and/or the CD83⁺ CCR7 ⁻ matureDCs of the invention transiently express CD40L polypeptide. Preferrably,CD40L is predominantly localized intracellularly, rather than on thecell surface. Most preferably, at least 60%, at least 70%, at least 80%or at least 90% of CD40L polypeptide is localized intracellularly.

In an alternative embodiment, the immature dendritic cells are signaledwith an effective amount of a TNF-α receptor agonist followed bysignaling with a CD40 agonist. Thus, the invention provides a method forpreparing mature dendritic cells (DCs), comprising sequentiallysignaling isolated immature dendritic cells with a first signalcomprising a tumor necrosis factor alpha receptor (TNF-αR) agonistfollowed by a second signal comprising a CD40 agonist, wherein saidsignaling is in the absence of an effective amount of IL-1β and/or IL-6.

For either embodiment (IFN-γR agonist or TNF-αR agonist as a firstsignal), the second CD40 agonist signal can be given to either CD83⁻CCR7 ⁻ iDCs, or to CD83⁺ CCR7 ⁻ mature DCs. In a preferred embodiment,the immature DCs and/or mature DCs are contacted with PGE₂. Preferablythe cells are contacted with PGE₂ at about the same time that theyreceive the first signal (an IFN-γR agonist or TNF-αR agonist). Inpreferred embodiments, GM-CSF and at least one of IL-4 or IL-13 ispresent in the medium at the time the dendritic cells receive the firstand second signals. In further embodiments, the method further comprisescontacting the immature dendritic cells, signaled dendritic cells,and/or CCR7⁺ dendritic cells with a NKT cell ligand that can activateCD1d-restricted NKT cells and consequently potentiate innate andadoptive immunity. In preferred embodiments, the NKT cell ligand is acompound selected from the group consisting of: α-galactosylceramides,α-glucosylceramides, α-6-deoxygalactosylceramides,α-6-deoxygalactofuranosylceramides, β-6-deoxygalactofuranosylceramides,β-arabinosylceramides, α-C-galactosylceramides andα-S-galactosylceramides. A preferred compound is theα-galactosylceramide known as KRN7000 ((2S, 3S,4R)-1-O—(alpha-D-galactopyranosyl)-2-(N-hexacosanoylamino)-1,3,4-octadecanetriol).

Agelasphins, disclosed in JP patent 3068910, are a class of compoundsoriginally discovered in a marine sponge which have anα-galactosylceramide (α-GalCer) structure and immunostimualting andanti-tumor activity. KRN7000 is a potent synthetic analog ofagelasphins, disclosed in U.S. Pat. No. 5,767,092, the contents of whichis incorporated by reference. Additional useful analogs of agelasphinsare disclosed U.S. Pat. No. 5,936,076, the contents of which isincorporated by reference. The structure of KRN7000 is shown below:

Glycosylceramide analogs of KRN7000 (e.g, α-galactosylceramides,α-glucosylceramides, α-6-deoxygalactosylceramides,α-6-deoxygalactofuranosylceramides, β-6-deoxygalactofuranosylceramides,β-arabinosylceramides) are disclosed in U.S. Pat. No. 5,849,716, thecontents of which is incorporated by reference. U.S. Pat. No. 5,780,441,the contents of which is incorporated by reference, disclosesoligosaccharide (di-, tri-, tetra-, penta-) derivatives of KRN7000.Methods for using KRN7000 and related analogs to produce KRN7000 antigenloaded DCs, and to activate human NKT cells are disclosed in U.S. SerNo. 09/721,768 and U.S. Pat. No. 6,531,453, the contents of each arespecifically incorporated by reference.

U.S. Pat. No. 5,936,076, the contents of which is incorporated byreference, discloses α-galactosylceramide compounds represented by thefollowing formula:

wherein the fatty acid chain, R represents:

where R₂ represents H or OH and X denotes an integer of 0-26 or Rrepresents −(CH₂)₇CH═CH(CH₂)₇CH₃ and

R₁ represents any one of the substituents defined by the following(a)-(c)

-   -   (a) —CH₂(CH₂)_(Y)CH₃    -   (b) —CH(OH)(CH₂)_(Y)CH₃    -   (c) —CH(OH)(CH₂)_(Y)CH(CH₃)₂    -   (d) —CH═CH(CH₂)_(Y)CH    -   (e) —CH(OH)(CH₂)_(Y)CH(CH₃)CH₂CH₃        Wherein Y denotes an integer 5-17.

WO 03/105769, U.S. 2004/0127429, the contents of which are incorporatedby reference, and Shimieg J. et al., (2003) J. Exp. Med. 198:1631-1641disclose the structure of α-C-glycolipids, where the oxygen atom onglycoside bond of α-glycosylceramides such as α-galactosylceramide andα-glucosylceramides is replaced by carbon atom. The structure of arepresentative compound is shown below.

WO 03/016326, the contents of which is incorporated, disclose KRN7000analogs with truncated ceramide such as “C4” or “OCH” having thefollowing structure:

U.S. Pat. No. 6,635,622, the contents of which is incorporated byreference, discloses α-C—, N, or S-Glycolipids, wherein the oxygen atomon glycoside bond of a galactosylceramide is replaced by—(CH₂)_(a)—CH═CH—(CH₂)_(a′)—, —(CH₂)_(a)—S(O)₀₋₂—CH₂—, or —NHCH₂—,wherein a and a′ each denote an integer of 0-5 and a+a′ is 5 or less.

In preferred embodiments, the IFN-γR agonist is IFNγ or a biologicallyactive fragment thereof. Preferably, the IFNγ is a mammalian IFNγ, mostpreferably a human IFNγ. The cDNA and amino acid sequence of human IFNγare shown in SEQ ID NOs: 5 and 6, respectively. Preferably, the IFNγ hasthe sequence shown in SEQ ID NO: 6, or a fragment thereof. In oneembodiment, the IFN-γR comprises a polypeptide having at least 80%sequence identity with SESQ ID NO: 6. Preferably, the IFN-γR agonist hasat least 85%, 90%, 95%, 97%, 98% or 99° A sequence identity with SEQ IDNO: 6. Methods for testing the activity of IFN-γR agonists are known tothose of skill in the art, and some of these methods are described belowImmature DCs can be signaled by adding an IFN-γR agonist the culturemedium, or by expressing the IFN-γR agonist in the dendritic cell. Inone embodiment, the DC is transfected with an mRNA encoding an IFN-γRagonist, such as SEQ ID NO: 6, or a biologically active fragmentthereof. Signaling would then occur upon translation of the mRNA withinthe dendritic cell. Most preferably, the IFN-γR agonist is added to theculture medium containing immature DCs. In a preferred embodiment, theculture medium further comprises PGE₂ and/or GM-CSF plus IL-4 or IL-13.

The receptor for IFN-γ has two subunits: IFN-γR1, the ligand-bindingchain (also known as the a chain) and IFN-γR2, the signal-transducingchain (also known as the β chain or accessory factor 1). These proteinsare encoded by separate genes (IFNGR1 and IFNGR2, respectively) that arelocated on different chromosomes. As the ligand-binding (or α) chainsinteract with IFN-γ they dimerise and become associated with twosignal-transducing (or β) chains. Receptor assembly leads to activationof the Janus kinases JAK1 and JAK2 and phosphorylation of a tyrosineresidue on the intracellular domain of IFN-γR1. This leads to therecruitment and phosphorylation of STAT1 (for ‘signal transducers andactivators of transcription’), which forms homodimers and translocatesto the nucleus to activate a wide range of IFN-γ-responsive genes. Aftersignaling, the ligand-binding chains are internalized and dissociate.The chains are then recycled to the cell surface. Bach et al. (1997)Ann. Rev. Immunol. 15, 563-591; and Lammas, Casanova and Kumararatne(2000) Clin Exp Immunol 121, 417-425. The crystal structure of thecomplex of human IFN-γ with the soluble, glycosylated extracellular partof IFN-γRα (sIFN-γRα) has been determined at 2.9 Å resolution usingmultiwavelength anomalous diffraction methods. Thiel et al. Structure8:927-936 (2000).

In one assay, INF-γ receptor agonists, such as IFN-γ decreaseNa⁺-K⁺-ATPase activity in a time- and concentration-dependent manner inhuman intestinal epithelial Caco-2 cells. Na⁺-K⁺-ATPase activity can bedetermined as the difference between total and ouabain-sensitive ATPase.Treatment with IFN-γ markedly increases the expression of total andphospho-STAT1, this being accompanied by activation of p38 MAPK. p38 MAPkinase activity can be analyzed by Western blotting using the p38 MAPkinase assay kit. Total and phosphorylated STAT1 protein levels weredetected using the PhosphoPlus® Stati. The transduction mechanisms setinto motion by IFN-γ involve the activation of PKC downstream STAT1phosphorylation and Raf-1, MEK, ERK2 and p38 MAPK pathways. See Magro etal., Br J Pharmacol advance online publication, Jul. 26, 2004; doi:10.1038/sj.bjp.0705895, the contents of which is incorporated byreference.

For the purpose of illustration, signaling with IFN-γ receptor agonists,TNF-α receptor agonists and/or CD40 agonists can be provided bycontacting a cell directly with IFN-γ polypeptides and/or proteinsand/or TNF-α polypeptides or proteins and/or CD40 agonists,respectively. Alternatively, signaling of a cell with IFN-γR agonists,TNF-αR agonists or CD40 agonists can occur upon translation of mRNAencoding such polypeptides or proteins within the dendritic cell. Thus,signaling occurs upon expression of IFN-γR agonist, TNF-αR agonist andCD40 agonist polypeptides and/or proteins.

The second signal used in the methods of the invention is a transientsignal with a CD40 agonist. Persistent expression of a CD40 agonistpolypeptide, such as constitutive expression of CD40L from a lentiviralvector as described by Koya et al., supra, is not considered transientexpression. The signal can be considered transient if the mediumcontaining a CD40 agonist is removed from the DCs, or if the DCs areloaded with an mRNA encoding a CD40 agonist. The CD40 agonist signal canalso be considered transient if the DCs are loaded/transfected with orwith an expression vector encoding a CD40 agonist, provided thateither: 1) the promoter driving CD40 agonist expression is notconstitutive in DCs, or 2) the expression vector does not integrate intothe DC genome or otherwise replicate in DCs.

In preferred embodiments, the CD40 agonist is a CD40L polypeptide or aCD40 agonistic antibody. In general, ligands that bind CD40 may act as aCD40 agonist. Applicants have demonstrated that administration of asecond signal comprising CD40L to the cells by transfection of immatureor mature DCs with CD40L mRNA produces subsequently modified DCs thatinduce immunostimulatory responses rather than immunosuppressive. In oneembodiment, CD40L mRNA transfected dendritic cells are cultured inmedium containing IFNγ (and preferentially PGE₂ as well) immediatelyafter transfection and prior to translation of the CD40L mRNA to producean effective amount of a CD40L signal. In this embodiment, although IFNγis added after transfection with CD40L mRNA, the dendritic cells receivethe IFNγ signal prior to the signal resulting upon translation of theCD40L mRNA. Thus, the order in which the agents are delivered to thecells is important only in that CD40L signaling must occur after IFN-γsignaling. As described in more detail below, the signaling of the DCscan occur in vivo or ex vivo, or alternatively one or more set may occurex vivo and the remaining steps of the method can occur in vivo.

In one embodiment, the CD40 agonist is an aptamer that binds CD40.Similarly, IFN-γ and TNF-α could be replaced by aptamers, antibodies,and the like, that have a similar biological activity. Most preferably,the CD40 agonist is delivered as mRNA encoding CD40L.

As used herein, “CD40 Ligand” (CD40L) shall encompass any polypeptide orprotein that specifically recognizes and activates the CD40 receptor andactivates its biological activity. The term includes transmembrane andsoluble forms of CD40L. In preferred embodiments, the CD40 agonist is amammalian CD40L, preferably a human CD40L. Alignments of the human andmouse cDNAs and proteins are shown in FIGS. 16 and 17, respectively. Ahuman CD40L cDNA and the corresponding amino acid sequence are shown inSEQ ID NOS: 1 and 2, respectively. The open reading frame for CD40L isrepresented by nucleotides 40 to 822 of SEQ ID NO.1, while the TGA stopcodon at position 823 to 825. In any of the CD40L polyneuclotidesequences of the invention, a silent mutation (a variant due to codondegeneracy) of the 102^(nd) codon in the CD40L sequence (nucleotides 343to 345 of SEQ ID NO: 1), changing the “AAA” codon to an “AAG” codon,both of which code for Lys may be used. Also useful in the methods ofthe invention are truncated CD40L (residues 47 to 261 of SEQ ID NO: 2,encoded by nucleotide residues 178 to 825 of SEQ ID NO: 1) and CD40Lfragments encoded by nucleotides 43 to 825 of SEQ ID NO: 1, 181 to 825of SEQ ID NO: 1, 193 to 825 of SEQ ID NO: 1, 376 to 825 of SEQ ID NO: 1,379 to 825 of SEQ ID NO: 1 and 400 to 825 of SEQ ID NO: 1. In preferredembodiments, the CD40L polypeptide is selected from the group consistingof: a) a polypeptide comprising SEQ ID NO: 2; b) a polypeptidecomprising amino acid residues 47 through 261 of SEQ ID NO: 2; c) apolypeptide comprising amino acid residues 51 through 261 of SEQ ID NO:2; d) a polypeptide comprising amino acid residues 120 through 261 ofSEQ ID NO: 2; e) a polypeptide comprising amino acid residues 113through 261 of SEQ ID NO: 2; f) a polypeptide comprising amino acidresidues 112 through 261 of SEQ ID NO: 2; g) a polypeptide comprisingSEQ ID NO: 10; h) a polypeptide comprising amino acid residues 35through 261 of SEQ ID NO: 2; i) a polypeptide comprising amino acidresidues 34 through 225 of SEQ ID NO: 2; j) a polypeptide comprisingamino acid residues 113 through 225 of SEQ ID NO: 2; k) a polypeptidecomprising amino acid residues 120 through 225 of SEQ ID NO: 2; and 1) afragment of the polypeptide of any of (a) through (k), wherein saidfragment binds CD40.

In various embodiments, the CD40L polypeptide is encoded by an mRNAcomprising a polynucleotide selected from the group consisting of: a) apolynucleotide of SEQ ID NO: 1; b) a polynucleotide comprisingnucleotides 40 to 822 of SEQ ID NO: 1; c) a polynucleotide comprisingnucleotides 178 to 822 of SEQ ID NO: 1; d) a polynucleotide comprisingnucleotides 190 to 822 of SEQ ID NO: 1; e) a polynucleotide comprisingnucleotides 397 to 822 of SEQ ID NO: 1; f) a polynucleotide comprisingnucleotides 376 to 822 of SEQ ID NO: 1; g) a polynucleotide of SEQ IDNO: 9; h) a polynucleotide of SEQ ID NO: 13; i) a polynucleotide havingat least 80% sequence identity with any polynucleotide of (a) through(h); j) a polynucleotide hybridizing under stringent conditions to anypolynucleotide of (a) through (h); and k) a polynucleotide of (a)through (j), further comprising a 3′ untranslated sequence selected fromthe group consisting of the nucleic acids of SEQ ID NO: 14, 15, 16, 17or 18, and/or a 5′ untranslated sequence selected from the groupconsisting of the nucleic acids of SEQ ID NO: 19, 20, 21, 22, or 23.Preferably, these RNAs are capped and polyadenylated.

Alternatively, the CD40L polypeptide is a polypeptide having at least77% sequence identity to a polypeptide selected from the groupconsisting of: a) a polypeptide comprising SEQ ID NO: 2; b) apolypeptide comprising amino acid residues 47 through 261 of SEQ ID NO:2; c) a polypeptide comprising amino acid residues 51 through 261 of SEQID NO: 2; d) a polypeptide comprising amino acid residues 120 through261 of SEQ ID NO: 2; e) a polypeptide comprising amino acid residues 113through 261 of SEQ ID NO: 2; f) a polypeptide comprising amino acidresidues 112 through 261 of SEQ ID NO: 2; g) a polypeptide comprisingSEQ ID NO: 10; h) a polypeptide comprising amino acid residues 35through 261 of SEQ ID NO: 2; i) a polypeptide comprising amino acidresidues 34 through 225 of SEQ ID NO: 2; j) a polypeptide comprisingamino acid residues 113 through 225 of SEQ ID NO: 2; k) a polypeptidecomprising amino acid residues 120 through 225 of SEQ ID NO: 2; and 1) afragment of the polypeptide of any of (a) through (k), wherein saidfragment binds CD40.

Most preferably, the CD40L polypeptide is the novel CD40L polypeptideprovided herein, consisting of, or consisting essentially of amino acidresidues 21 to 261 of SEQ ID NO: 2, or a polypeptide having at least80%, more preferably at least 85%, 90%, 95%, 96%, 97%, 98% or mostpreferably at least 99% homology thereto. Preferably, the CD40Lpolypeptide is encoded by an RNA corresponding to the cDNA of SEQ ID NO:30 or SEQ ID NO: 33, or to variants which differ do to codon degeneracy.As used herein, an RNA corresponding to a cDNA sequence refers to an RNAsequence having the same sequence as the cDNA sequence, except that thenucleotides are ribonucleotides instead of deoxyribonucletides, asthymine (T) base in DNA is replaced by uracil (U) base in RNA.Preferably, the RNAs are capped and polyadenylated. Accordingly, theinvention provides a CD40L polypeptide which consists of or consistingessentially of amino acid residues 21-261 of SEQ ID NO: 2. In anotheraspect, the invention provides a nucleic acid encoding a CD40Lpolypeptide which consists of or consisting essentially of amino acidresidues 21-261 of SEQ ID NO: 2. Dendritic cells transfected with suchnucleic acids are also provided, as well as vaccines comprising suchdendritic cells. Preferably, the dendritic cells are transientlytransfected with the RNA encoding these novel CD40L polypeptides of theinvention.

In another aspect, the invention provides a method for preparing maturedendritic cells (DCs), comprising the sequential steps of: (a) signalingisolated immature dendritic cells (iDCs) with a first signal comprisingan interferon gamma receptor (IFN-γR) agonist, and optionally, a TNF-αRagonist, to produce IFN-γR agonist signaled dendritic cells; and (b)signaling said IFN-γR agonist signaled dendritic cells with a secondtransient signal comprising an effective amount of a CD40L polypeptideto produce CCR7⁺ mature dendritic cells; wherein the CD40L polypeptideconsists essentially of amino acid residues 21-261 of SEQ ID NO: 2 or apolypeptide having at least 80% sequence identity to amino acid residues21-261 of SEQ ID NO: 2.

CD40 was first characterized as a receptor expressed on B lymphocytes.Schonbeck and Libby (2001) Cell Mol. Life Sci. 58:4. It was laterdiscovered that engagement of B-cell CD40 with CD40L expressed onactivated T-cells is essential for T-cell dependent B-cell activation(i.e. proliferation, immunoglobulin secretion, and class switching). Itwas subsequently revealed that functional CD40 is expressed on a varietyof cell types other than B-cells, including hematopoietic progenitorcells, T lymphocytes, basophils, eosinophils, monocytes/macrophages,dendritic cells, epithelial cells, endothelial cells, smooth musclecells, keratinocytes, fibroblasts and carcinomas. Schonbeck and Libby(2001) supra.

The CD40 Ligand was cloned in 1993 and reported by Gauchat, et al.(1993) FEBS Lett. 315:259. Graf et al. mapped it to chromosomeXq26.3-q27.1 (Graf, et al. (992) Eur. J. Immunol. 22: 3191-3194).Shorter soluble forms of the cell-associated full-length 39 kDa form ofCD40 Ligand have been described with molecular weights of 33, and 18kDa. Graf, et al. (1995) Eur. J. Immunol. 25: 1749; Ludewig, et al.(1996) Eur. J. Immunol. 26: 3137; Wykes, et al. (1998) Eur. J. Immunol.28:548. The 18 kDa soluble form generated via intracellular proteolyticcleavage, which lacks the cytoplasmic tail, the transmembrane region andparts of the extracellular domain, but conserves the CD40 binding domainretains the ability to bind to CD40 receptor and therefore is an exampleof a CD40 receptor signaling agent. Graf, et al. (1995) supra.

U.S. Pat. No. 5,981,724 discloses DNA sequences encoding human CD40Ligand (CD40L) as well as vectors, and transformed host cells for thepurpose of producing CD40L polypeptides. U.S. Pat. No. 5,962,406discloses DNA sequences encoding soluble forms of human CD40L.

Exemplary sequences of mammalian homologs to CD40L have the followingGenbank accession numbers: NM_204733 (Gallus gallus (chicken)); DQ054533(Ovis aries (sheep)); Z48469 (Bos taurus (cow)); AY333790 (Canisfamiliaris (dog)); Macaca nemestrina (pig-tailed macaque)); AF344844(Callithrix jacchus (white-tufted-ear marmoset)); AF34481 (Cercicebustorquatus atys (sooty mangabey)); AF344860 (Aotus trivirgatus(douroucouli)); AF344859 Macaca mulatta (rhesus monkey)); AF116582(Rattus nevegicus (Norway rat)); and AF079105 (Felus catus (cat)).

The CD40 receptor can also be activated by use of CD40 agonistantibodies, antibody fragments, derivatives and variants thereof. CD40agonist antibodies can be purchased from commercial vendors such asMabtech (Nacka, Sweden). Examples and methods to generate these agentsare also provided infra. The literature also provides examples of CD40agonist antibodies and antibody fragments. See, e.g., Osada, et al.(2002) 25(2): 176 and Ledbetter, J. A. et al. (1997) Crit. Reviews inImmunol. 17:427.

As noted above, the agent having the biological activity of CD40L can bea polypeptide translated from an exogenous polynucleotide (mRNA or DNA)encoding CD40L. For example, the CD40L mRNA has the sequence of SEQ IDNO.: 1 or SEQ ID NO.: 3. Alternatively, the cells are signaled with aneffective amount of CD40L protein and/or polypeptide, for example, thosehaving the sequence of SEQ ID NO.: 2 or SEQ ID NO.: 4. Modified CD40Lcan also be used in the methods of this invention. For example, CD40Lincludes those molecules that have been altered through addition,subtraction, or substitution, either conservatively ornon-conservatively, of any number of amino acids, provided that theresulting protein binds CD40 on the surface of DC. A “conservativealteration” is one that results in an alternative amino acid of similarcharge density, hydrophilicity or hydrophobicity, size, and/orconfiguration (e.g., Val for Ile). In comparison, a “nonconservativealteration” is one that results in an alternative amino acid ofdiffering charge density, hydrophilicity or hydrophobicity, size and/orconfiguration (e.g., Val for Phe). The means of making suchmodifications are well-known in the art and also can be accomplished bymeans of commercially available kits and vectors (for example, thoseavailable from New England Biolabs, Inc., Beverly, Mass.; Clontech, PaloAlto, Calif.).

When the agents are delivered as polynucleotides or genes encoding theagents, an effective amount of the polynucleotide can be replicated byany method known in the art. PCR technology is one means to replicateDNA and is the subject matter of U.S. Pat. Nos. 4,683,195; 4,800,159;4,754,065; and 4,683,202 and described in PCR: THE POLYMERASE CHAINREACTION (Mullis et al. eds, Birkhauser Press, Boston (1994)) andreferences cited therein. Additional methods to generate polynucleotidesare provided infra.

In embodiments of the invention, wherein immature dendritic cells arestimulated with an agonist of TNF-α receptor, followed by stimulationwith a CD40 agonist, the method is performed in the absence of aneffective amount of interleukin 1-beta (IL-1β) and or interleukin 6(IL-6). Methods for detecting the presence of proteins such as IL-1β andIL-6 are known in the art.

One of skill in the art can determine when the object of the method hasbeen met by sampling a cell or small population of DCs from thepopulation for the presence of mature DCs expressing CD40L mRNA and/orCD40L polypeptide. In a further aspect, the mature CD83⁺ CCR7⁺ DCs ofthe invention express interleukin 12 (IL-12) p35 protein. In a furtheraspect, mature CD83⁺ CCR7⁺ DCs express IL-12 p70 protein, and/or expresslimited IL-10 (not more than 500 pg/ml/10⁶ DCs).

The steps of the method can be practiced in vivo or ex vivo. Whenpracticed ex vivo, the method can be practiced in an open or closedsystem. Methods and systems for culturing and enriching cell populationsare known in the art. See, examples 1 and 2 of U.S. Patent PublicationNo. 2004/0072347. See also U.S. Patent Publication No. 2003/0235908,which describes closed systems for cell expansion.

In a further aspect, of this invention, the above method is modified bythe addition of delivering to the immature or mature DCs an effectiveamount of an antigen which will be then be processed and presented bythe mature DCs. Thus, the methods of the invention further compriseintroducing into iDCs, signaled DCs or CCR7⁺ mature DCs one or moreantigens or a polynucleotide(s) encoding one or more antigens to producean antigen-loaded CCR7⁺ mature DCs. The antigen or antigen-encodingpolynucleotide can be introduced prior to said first signal.Alternatively, the antigen or antigen-encoding polynucleotide isdelivered subsequent to said first signal and prior to said secondsignal. In another embodiment, the antigen or polynucleotide isdelivered subsequent to said second signal or substantially concurrentwith said second signal.

For example, antigens include, but are not limited to, pathogens,pathogen lysates, pathogen extracts, pathogen polypeptides, viralparticles, bacteria, proteins, polypeptides, cancer cells, cancer celllysates, cancer cell extracts, cancer cell specific polypeptides.Antigens can be naturally occurring or recombinantly produced. Theimmunogens can be delivered to the cells as polypeptides, proteins or asnucleic acids using methods known in the art which are briefly describedinfra. Preferably, one or more polynucleotides encoding one or moreantigens are introduced into the iDCs, signaled DCs or CCR7⁺ mature DCs.The polynucleotide can be introduced into the DCs by methods known tothose of skill in the art. In a preferred embodiment, the polynucleotideis introduced by electroporation. Most preferably, the polynucleotide isan mRNA. In preferred embodiments, the antigen or antigen encoding mRNAis introduced together with an mRNA encoding a CD40 agonist orsubstantially concurrent with CD40 agonist signaling.

The methods can be further modified by contacting the cell with aneffective amount of a cytokine or co-stimulatory molecule, e.g., GM-CSF,IL-4 and PGE₂. In embodiments where the immature DCs are signaled with aTNFαR agonist followed by signaling with CD40 agonist, effective amountsof IL-1β and/or IL-6 are specifically excluded from the culture.

The antigen may be delivered in its “natural” form in that no humanintervention was involved in preparing the antigen or inducing it toenter the environment in which it encounters the APC. Alternatively oradditionally, the antigen may comprise a crude preparation, for exampleof the type that is commonly administered in a conventional allergy shotor in a tumor lysate. The antigen may alternatively be substantiallypurified, e.g., at least about 90% pure.

Where the antigen is a peptide, it may be generated, for example, byproteolytic cleavage of isolated proteins. Any of a variety of cleavageagents may be utilized including, but not limited to, pepsin, cyanogenbromide, trypsin, chymotrypsin, etc. Alternatively, peptides may bechemically synthesized, preferably on an automated synthesizer such asis available in the art. Also, recombinant techniques may be employed tocreate a nucleic acid encoding the peptide of interest, and to expressthat peptide under desired conditions.

The antigen can alternatively have a structure that is distinct from anynaturally-occurring compound. In certain embodiments of the invention,the antigen is a “modified antigen” in that the antigen has a structurethat is substantially identical to that of a naturally-occurring antigenbut that includes one or more deviations from the precise structure ofthe naturally-occurring compound. For instance, where thenaturally-occurring antigen is a protein or polypeptide antigen, amodified antigen as compared with that protein or polypeptide antigenwould have an amino acid sequence that differs from that of thenaturally-occurring antigen in the addition, substitution, or deletionof one or more amino acids, and/or would include one or more amino acidsthat differ from the corresponding amino acid in the naturally-occurringantigen by the addition, substitution, or deletion of one or morechemical moieties covalently linked to the amino acid. In one aspect,the naturally-occurring and modified antigens share at least one regionof at least 5 amino acids that are at least approximately 75% identical.Those of ordinary skill in the art will appreciate that, in comparingtwo amino acid sequences to determine the extent of their identity, thespacing between stretches (i.e., regions of at least two) of identicalamino acids need not always be precisely preserved. Naturally-occurringand modified protein or polypeptide antigens can show at leastapproximately 80% identity, more alternatively 85%_(,) 90%_(,) 95%, orgreater than 99% identity in amino acid sequence for at least one regionof at least 5 amino acids. Often, it may be useful for a much longerregion (e.g., 10, 20, 50, or 100 or more amino acids) of amino acidsequence to show the designated degree of identity.

In preferred embodiments, the antigen is delivered as a polynucleotideor gene encoding the antigen, so that expression of the gene results inantigen production either in the individual being treated (whendelivered in vivo) or the cell culture system (when delivered in vitro).Techniques for generating nucleic acids including an expressible gene,and for introducing such nucleic acids into an expression system inwhich any protein encoded by the expressible gene will be produced areknown in the art and briefly described infra. Preferrably, an mRNAencoding the antigen is introduced into the DC.

In one embodiment, the immunogen is delivered prior to said firstsignal, wherein the first signal is an IFNγR agonist or TNF-αR.Alternatively, the immunogen is delivered subsequent to said firstsignal and prior to said second signal, or the immunogen is deliveredsubsequent to said second signal. In another embodiment, the immunogenis delivered substantially concurrent with said second signal.

The amount of antigen to be employed in any particular composition orapplication will depend on the nature of the particular antigen and ofthe application for which it is being used, as will readily beappreciated by those of skill in the art.

The antigen-loaded dendritic cells are useful for raising an immuneresponse to the antigen(s). Thus, in one aspect, the invention providesa method of raising an immune response in a subject comprisingadministering to the subject an effective amount of the immunogen loadedCCR7¹ mature DCs. The loaded DCs may be allogeneic or autologous to thesubject.

The invention further provides a method of stimulating immune effectorcells, comprising culturing said cells in the presence of an antigenloaded CCR7¹ mature DCs produced by the methods of invention to producestimulated immune effector cells. In another embodiment, the inventionprovides a method of enhancing immunity in a subject comprisingadministering to the subject an effective amount of such stimulatedimmune effector cells.

In a further aspect of this invention, an effective amount of a cytokineand/or co-stimulatory molecule is delivered to the cells or patient, invitro or in vivo. These agents can be delivered as polypeptides,proteins or alternatively, as the polynucleotides or genes encodingthem. Cytokines, co-stimulatory molecules and chemokines can be providedas impure preparations (e.g., isolates of cells expressing a cytokinegene, either endogenous or exogenous to the cell) or in a “purified”form. Purified preparations are preferably at least about 90% pure, oralternatively, at least about 95% pure, or yet further, at least about99% pure. Alternatively, genes encoding the cytokines or inducing agentsmay be provided, so that gene expression results in cytokine or inducingagent production either in the individual being treated or in anotherexpression system (e.g., an in vitro transcription/translation system ora host cell) from which expressed cytokine or inducing agent can beobtained for administration to the individual.

Where both cytokine and antigen are to be delivered to an individual,they may be provided together or separately. When they are delivered aspolypeptides or proteins, they can be delivered in a commonencapsulation device or by means of physical association such ascovalent linkage, hydrogen bonding, hydrophobic interaction, van derWaals interaction, etc. In an alternative embodiment, the compounds areprovided together, genes encoding both are provided. For example, genesfor both may be provided as part of the same nucleic acid molecule. Insome embodiments, this nucleic acid molecule may be prepared so thatboth factors are expressed from a single contiguous polynucleotide, as afusion protein in which the cytokine and the antigen are covalentlylinked to one another via a peptide bond. Alternatively or additionally,the genes may be linked to the same or equivalent control sequences, sothat both genes become expressed within the individual in response tothe same stimuli. A wide variety of different control sequences, activein different host cells under different conditions are known in the art.These control sequences, including constitutive control sequences,inducible control sequences, and repressible control sequences, can beused in accordance with the present invention, though inducible orrepressible sequences are particularly preferred for applications inwhich additional control over the timing of gene expression is desired.

It is appreciated by those of skill in the art that administration ofcytokine and/or antigen may optionally be combined with theadministration of any other desired immune system modulatory factor suchas, for example, an adjuvant or other immunomodulatory compound.

Antigens can also be delivered in the form of polynucleotides or genesencoding the antigens. The antigens can also be modified by linking aportion of sequence from a first polypeptide (e.g., a first antigen) toa portion of sequence from a second polypeptide (e.g., a second antigen,a signal sequence, a transmembrane domain, a purification handle, etc.)by means of a peptide bond. Those of ordinary skill in the art willappreciate the diversity of such fusion proteins for use in accordancewith the present invention. Recombinant techniques further allow for theready modification of the amino acid sequence of polypeptide or proteinantigens, by substitution, deletion, addition, or inversion of aminoacid sequences.

Where the immunogen is a fragment of an antigen, it may be generated,for example, by proteolytic cleavage of isolated proteins. Any of avariety of cleavage agents may be utilized including, but not limitedto, pepsin, cyanogen bromide, trypsin, chymotrypsin, etc. Alternatively,peptides may be chemically synthesized, preferably on an automatedsynthesizer such as is available in the art (see, for example, Stewartet al., Solid Phase Peptide Synthesis, 2d. Ed., Pierce Chemical Co.,1984. Also, recombinant techniques may be employed to create a nucleicacid encoding the peptide of interest, and to express that peptide underdesired conditions (e.g., in a host cell or an in vitro expressionsystem from which it can readily be purified).

In preferred embodiments, the antigen is from a cancer cell or apathogen. Preferably, the neoplastic cell is a renal cancer cell, amultiple myeloma cell or a melanoma cell. Preferred pathogens are HIVand HCV. In preferred embodiments, the antigen is delivered to theantigen presenting cell in the form of RNA isolated or derived from aneoplastic cell or a pathogen. Methods for RT-PCR of RNA extracted fromany cell (e.g., a neoplastic cell or pathogen cell), and in vitrotranscription are disclosed in copending PCT/US05/32710 and U.S.provisional patent application No. 60/525,076, the contents of which areincorporated by reference.

The antigen employed in accordance with the present invention may be anaturally-occurring compound or may alternatively have a structure thatis distinct from any naturally-occurring compound. In certainembodiments of the invention, the antigen is a “modified antigen” inthat the antigen has a structure that is substantially identical to thatof a naturally-occurring antigen but that includes one or moredeviations from the precise structure of the naturally-occurringcompound.

Also provided by this invention are the enriched populations of matureDCs prepared by any of the methods described herein. Mature DCs preparedby the methods of the invention have enhanced immunostimulatorycharacteristics. In another aspect, the invention provides a method forstoring an enriched population of mature DCs, comprising contacting anenriched dendritic cell population of the invention with a suitablecryopreservative under suitable conditions.

The compositions described herein are useful to raise an immune responsein a subject by administering to the subject an effective amount of theenriched population of cells, e.g., DCs, modified DCs, or educatedimmune effector cells. The cells can be allogeneic or autologous. Theycan be administered to a subject to raise or induce an immune responsein a subject comprising administering to the subject an effective amountof the enriched populations as described above. The cells can beallogeneic or autologous to the subject. They can also be used toeducate immune effector cells such as T cells by culturing the immuneeffector cell in the presence and at the expense of a mature DC of thisinvention. The educated effector cells can also be used to enhanceimmunity in a subject by delivering to the subject an effective amountof these cells.

Methods for Generating and Delivering Polynucleotides

Certain embodiments of this invention require the use ofpolynucleotides. These can be generated and replicated using any methodknown in the art, e.g., one of skill in the art can use the sequencesprovided herein and a commercial DNA synthesizer to replicate the DNA.Alternatively, they can be obtained by providing the linear sequence ofthe polynucleotide, appropriate primer molecules, chemicals such asenzymes and instructions for their replication and chemicallyreplicating or linking the nucleotides in the proper orientation toobtain the polynucleotides. In a separate embodiment, thesepolynucleotides are further isolated. Still further, one of skill in theart can insert the polynucleotide into a suitable replication vector andinsert the vector into a suitable host cell (prokaryotic or eukaryotic)for replication and amplification. The DNA so amplified can be isolatedfrom the cell by methods well known to those of skill in the art. Aprocess for obtaining polynucleotides by this method is further providedherein as well as the polynucleotides so obtained.

In one embodiment, the agent (e.g., CD40L) is delivered as mRNA. RNA canbe obtained by first inserting a DNA polynucleotide into a suitable hostcell or preferably, by in vitro transcription. The DNA can be insertedby any appropriate method, e.g., by the use of an appropriate genedelivery vehicle (e.g., liposome, plasmid or vector) or byelectroporation. When the cell replicates and the DNA is transcribedinto RNA; the RNA can then be isolated using methods well known to thoseof skill in the art, for example, as set forth in Sambrook et al. (1989)supra. For instance, mRNA can be isolated using various lytic enzymes orchemical solutions according to the procedures set forth in Sambrook, etal. (1989) supra or extracted by nucleic-acid-binding resins followingthe accompanying instructions provided by the manufacturer.

In preferred embodiments the CD40L expression cassette contains apromoter suitable for in vitro transcription, such as the T7 promoter orSP6 promoter. Preferably, the in vitro transcribed CD40L or CD40 agonistmRNA is optimized for stability and efficiency of translation. Forexample, SEQ ID NO: 13 represents an optimized CD40L mRNA, wherein ATGcodons in the 5′ untranslated region have been altered to avoidincorrect initiation of translation.

mRNA stability and/or translational efficiency can also be increased byincluding 3′UTRs and or 5′UTRs in the mRNA. Preferred examples of 3′UTRsinclude those from human CD40, β-actin and rotavirus gene 6. Preferredexamples of 5′UTRs include CD40L, and the translational enhancers in the5′UTRs of Hsp70, VEGF, spleen necrosis virus RU5, and tobacco etchvirus.

For example, CD40L expression is normally regulated in part by3′UTR-mediated mRNA instability, and therefore a large portion of theCD40L 3′UTR is not included in the current CD40L mRNA. CD40L is notnormally expressed in DCs. In contrast, the CD40 Receptor is expressedin DCs and there is no evidence in the literature to indicate that itsexpression is regulated post-transcriptionally, particularly at thelevel of mRNA stability. Including the CD40 Receptor 3′UTR (SEQ ID NO:14, or an active fragment thereof) at the 3′ end or region of the CD40LmRNA would give the RNA 3′ untranslated sequence similar to naturallyoccurring CD40 messages without imparting any unwanted regulatoryactivity.

Beta-Actin is an abundantly expressed gene in human non-muscle cells.The human beta-actin promoter has been widely used to drive geneexpression in mammalian cell lines and transgenic mice. Inclusion of thebeta-actin 3′UTR plus flanking region has been demonstrated to furtherincrease the level of mRNA accumulation from gene expression constructscontaining the beta-actin promoter.

Qin and Gunning (1997) Journal of Biochemical and Biophysical Methods 36pp. 63-72. SEQ ID NO: 15 represents the untranslated region of the finalexon of the human beta-actin 3′ UTR. SEQ ID NO: 16 shows the minimalregion of this 3′UTR.

The 3′UTR of the simian rotavirus gene 6 (SEQ ID NO: 17) mRNA functionsas an enhancer of translation in its capped, non-polyadenylated viraltranscript. The 3′UTR has also been shown to enhance translation of aheterologous reporter mRNA in Rabbit reticulocyte lysates. Yang et. al.,2004 Archives of Virology 149:303-321. The minimal functional element ofthis 3′UTR is shown in SEQ ID NO: 18

The 5′ UTR of the human hsp70 gene (SEQ ID NO: 19) has been shown toincrease translation of reporter mRNAs in the absence of stressinduction and without dramatically influencing the message stability.Enhancer function has been demonstrated in a number of human cell lines.Vivinus, et al., 2001 European Journal of Biochemistry 268:1908-1917.

The mouse VEGF 5′ UTR (SEQ ID NO: 20) enhances translation of amonocistronic reporter RNA and also has IRES (Internal Ribosome EntrySite) activity. Its enhancer activity has been demonstrated in rat,hamster and human cell lines. The full length 5′UTR is 1014 nucleotides,but a 163 nucleotide mutant version (SEQ ID NO: 21) was shown to be moreactive. Stein et al., 1998 Molecular and Cellular Biology 18:3112-3119.

The Spleen Necrosis Virus (SNV) is an avian retrovirus. The RU5 regionof the viral 5′ LTR (SEQ ID NO: 22) stimulates translation efficiency ofa non-viral reporter RNA in human 293 cells. Roberts and Boris-Lawrie(2000) Journal of Virology 74:8111-8118.

The 143 nucleotide 5′ leader of the tobacco etch virus RNA (SEQ ID NO:23) promotes cap-independent translation of reporter mRNAs in plant andanimal cell lines. Although the leader sequence does not further enhancethe translation of capped transcripts, the cap-independent CD40Lexpression in dendritic cells is a very attractive alternative to invitro capping. Gallie et al. (1995) Gene 165:233-238. Niepel and Gallie(1999) Journal of Virology 73:9080-9088. Gallie, Journal of Virology(2001) 75:12141-12152.

Human globin mRNAs are highly stable in erythrocyte progenitor cellswith half-lives ranging from 16 to 20 hours. The cis-acting elementsrequired for α- and β-globin mRNA stability have been well defined andare located in the 3′ untranslated region of each meassage (Holcik andLiebehaber, 1997 PNAS 94 2410-2414; and Yu and Russell, 2001, Molecularand Cellular Biology 21(17) 5879-5888). The sequence of the humanα-globin 3′UTR is shown in SEQ ID NO: 27. The sequence of the humanβ-globin 3′UTR is shown in SEQ ID NO: 28. The sequence of the humanβ-globin 3′UTR, minus Purine-Rich Element 3 is shown in SEQ ID NO: 29.

Dendritic cells can be transfected with nucleic acids by methods knownin the art, which include, but are not limited to calcium phosphateprecipitation, microinjection or electroporation. They can be addedalone or in combination with a suitable carrier, e.g., apharmaceutically acceptable carrier such as phosphate buffered saline.Alternatively or additionally, the nucleic acid can be incorporated intoan expression or insertion vector for incorporation into the cells.Vectors that contain both a promoter and a cloning site into which apolynucleotide can be operatively linked are known in the art. Suchvectors are capable of transcribing RNA in vitro or in vivo, and arecommercially available from sources such as Stratagene (La Jolla,Calif.) and Promega Biotech (Madison, Wis.). In order to optimizeexpression and/or in vitro transcription, it may be necessary to remove,add or alter 5′ and/or 3′ untranslated portions of the clones toeliminate extra, potential inappropriate alternative translationinitiation codons or other sequences that may interfere with or reduceexpression, either at the level of transcription or translation.Alternatively, consensus ribosome binding sites can be insertedimmediately 5′ of the start codon to enhance expression. Examples ofvectors are viruses, such as baculovirus and retrovirus, bacteriophage,adenovirus, adeno-associated virus, cosmid, plasmid, fungal vectors andother recombination vehicles typically used in the art which have beendescribed for expression in a variety of eukaryotic and prokaryotichosts, and may be used for gene therapy as well as for simple proteinexpression.

Among these are several non-viral vectors, including DNA/liposomecomplexes, and targeted viral protein DNA complexes. To enhance deliveryto a cell, the nucleic acid or proteins of this invention can beconjugated to antibodies or binding fragments thereof which bind cellsurface antigens. Liposomes that also comprise a targeting antibody orfragment thereof can be used in the methods of this invention. Thisinvention also provides the targeting complexes for use in the methodsdisclosed herein.

Polynucleotides are inserted into vector genomes using methods known inthe art. For example, insert and vector DNA can be contacted, undersuitable conditions, with a restriction enzyme to create complementaryends on each molecule that can pair with each other and be joinedtogether with a ligase. Alternatively, synthetic nucleic acid linkerscan be ligated to the termini of restricted polynucleotide. Thesesynthetic linkers contain nucleic acid sequences that correspond to aparticular restriction site in the vector DNA. Additionally, anoligonucleotide containing a termination codon and an appropriaterestriction site can be ligated for insertion into a vector containing,for example, some or all of the following: a selectable marker gene,such as the neomycin gene for selection of stable or transienttransfectants in mammalian cells; enhancer/promoter sequences from theimmediate early gene of human CMV for high levels of transcription;transcription termination and RNA processing signals from SV40 for mRNAstability; SV40 polyoma origins of replication and Co1E1 for properepisomal replication; versatile multiple cloning sites; and T7 and SP6RNA promoters for in vitro transcription of sense and antisense RNA.Other means are known and available in the art.

Preparation and Isolation of Proteins and Polypeptides

Polypeptides and proteins are necessary components of various methods ofthis invention. The proteins and polypeptides can be obtained bychemical synthesis using a commercially available automated peptidesynthesizer such as those manufactured by Perkin Elmer/AppliedBiosystems, Inc., Model 430A or 431A, Foster City, Calif., USA. Thesynthesized protein or polypeptide can be precipitated and furtherpurified, for example by high performance liquid chromatography (HPLC).Alternatively, the proteins and polypeptides can be obtained by knownrecombinant methods as described herein using the host cell and vectorsystems described below.

It is well know to those skilled in the art that modifications can bemade to any peptide to provide it with altered properties. As usedherein the term “amino acid” refers to either natural and/or unnaturalor synthetic amino acids, including glycine and both the D and L opticalisomers, and amino acid analogs and peptidomimetics. A peptide of threeor more amino acids is commonly called an oligopeptide if the peptidechain is short. If the peptide chain is long, the peptide is commonlycalled a polypeptide or a protein. Peptides for use in this inventioncan be modified to include unnatural amino acids. Thus, the peptides maycomprise D-amino acids, a combination of D- and L-amino acids, andvarious “designer” amino acids (e.g., β-methyl amino acids, C-α-methylamino acids, and N-α-methyl amino acids, etc.) to convey specialproperties to peptides. Additionally, by assigning specific amino acidsat specific coupling steps, peptides with α-helices β turns, β sheets,γ-turns, and cyclic peptides can be generated. In a further embodiment,subunits of peptides that confer useful chemical and structuralproperties will be chosen. For example, peptides comprising D-aminoacids may be resistant to L-amino acid-specific proteases in vivo.Modified compounds with D-amino acids may be synthesized with the aminoacids aligned in reverse order to produce the peptides of the inventionas retro-inverso peptides. In addition, the present invention envisionspreparing peptides that have better defined structural properties, andthe use of peptidomimetics, and peptidomimetic bonds, such as esterbonds, to prepare peptides with novel properties. In another embodiment,a peptide may be generated that incorporates a reduced peptide bond,i.e., R₁—CH₂NH—R₂, where R₁, and R₂ are amino acid residues orsequences. A reduced peptide bond may be introduced as a dipeptidesubunit. Such a molecule would be resistant to peptide bond hydrolysis,e.g., protease activity. Such molecules would provide peptides withunique function and activity, such as extended half-lives in vivo due toresistance to metabolic breakdown, or protease activity. Furthermore, itis well known that in certain systems constrained peptides show enhancedfunctional activity (Hruby (1982) Life Sciences 31:189-199 and Hruby etal. (1990) Biochem 3. 268:249-262); the present invention provides amethod to produce a constrained peptide that incorporates randomsequences at all other positions.

Methods for Isolating Stem Cells

Many methods are known in the art for the isolation and expansion ofCD34+ stem cells for in vitro expansion and differentiation intodendritic cells. See for example, U.S. Pat. No. 5,199,942, the contentsof which is incorporated by reference. The following descriptions arefor the purpose of illustration only and in no way are intended to limitthe scope of the invention.

CD34⁺ stem cells can be isolated from bone marrow cells or by panningthe bone marrow cells or other sources with antibodies which bindunwanted cells, such as CD4⁺ and CD8⁺ (T cells), CD45⁺ (panB cells) andGR-1 For a detailed description of this protocol see, Inaba, et al.(1992) 3. Exp. Med. 176:1693-1702. Human CD34⁺ cells can be obtainedfrom a variety of sources, including cord blood, bone marrow explants,and mobilized peripheral blood. Purification of CD34⁺ cells can beaccomplished by antibody affinity procedures. See, for example, Paczesnyet al. (2004) 3 Exp Med. 199: 1503-11; Ho, et al. (1995) Stem Cells 13(suppl. 3): 100-105; Brenner (1993) Journal of Hematotherapy 2:7-17; andYu, et al. (1995) PNAS 92:699-703.

Differentiating Stem Cells into Immature Dendritic Cells

CD34⁺ stem cells can be differentiated into dendritic cells byincubating the cells with the appropriate cytokines. Inaba et al. (1994)supra, described the in vitro differentiation of murine stem cells intodendritic cells by incubating the stem cells with murine GM-CSF. Inbrief, isolated stem cells are incubated with between 1 and 200 ng/mlmurine GM-CSF, and preferably about 20 ng/ml GM-CSF in standard RPMIgrowth medium. The media is changed with fresh media about once everyother day. After approcimatedly 5-7 days in culture, a large percentageof cells are dendritic, as assessed by expression of surface markers andmorphology. Dendritic cells are isolated by florescence activated cellsorting (FACS) or by other standard methods.

Murine CD34⁺ stem cells can be differentiated into dendritic cells byculturing the cells with murine GM-CSF. Typically, the concentration ofGM-CSF in culture is at least about 0.2 ng/ml, and preferably at leastabout 1 ng/ml. Often the range will be between about 20 ng/ml and 200ng/ml. In many preferred embodiments, the dose will be about 100 ng/ml.IL-4 is optionally added in similar ranges for making murine DCs.

Human CD34¹ hematopoietic stem cells are preferably differentiated invitro by culturing the cells with human GM-CSF and TNF-α. See forexample, Szabolcs, et al. (1995) 154:5851-5861. Human GM-CSF is used insimilar ranges, and TNF-α can also added to facilitate differentiation.TNF-α is also typically added in about the same ranges. Optionally, SCFor other proliferation ligand (e.g., F1t3) is added in similar doseranges to differentiate human DCs.

As is apparent to those of skill in the art, dose ranges fordifferentiating stem cells and monocytes into dendritic cells areapproximate. Different suppliers and different lots of cytokine from thesame supplier vary in the activity of the cytokine. One of skill caneasily titrate each cytokine which is used to determine the optimal dosefor any particular cytokine.

Differentiation of Monocytes into Dendritic Cells

DCs can be generated from frequent, but non-proliferating CD14⁺precursors (monocytes) in peripheral blood by culture in mediumcontaining GM-CSF and IL-4 or GM-CSF and IL-13 (see, e.g., WO 97/29182).This method is described in Sallusto and Lanzavecchia (1994) J. Exp.Med. 179:1109 and Romani et al. (1994) J. Exp. Med. 180:83. Briefly, CD14⁺ precursors are abundant so that pretreatment of patients withcytokines such as G-CSF (used to increase CD34¹ cells and more committedprecursors in peripheral blood) is reported to be unnecessary in mostcases (Romani et al. (1996) J. Immunol. Methods 196:137). Others havereported that DCs generated by this approach appear rather homogenousand can be produced in an immature state or fully differentiated ormature. It was shown that it is possible to avoid non-human proteinssuch as FCS (fetal calf serum), and to obtain fully and irreversiblymature and stable DCs by using autologous monocyte conditioned medium asmaturation stimulus (Romani et al. (1996) Immunol. Methods 196:137;Bender et al. (1996) J. Immunol. Methods 196:121). However, in contrastto the instant invention, these studies did not result in mature DChaving increased levels of IL-12 and/or decreased levels of IL-10.

Antigen Loading

Methods of loading dendritic cells with antigens are known to those ofskill in the art. In one embodiment, the dendritic cells are cultured inmedium containing the antigen. The DCs then take up and process theantigen on the cell surface in association with MHC molecules.Preferably, the DCs are loaded with antigen by transfection with anucleic acid encoding the antigen. Methods of transfecting DCs are knownto those of skill in the art.

Isolation of and Expansion of T Cells

In some methods of this invention, T cells are isolated from mammals sothat they can be educated (or activated) by the mature, modified DC invitro. In one method, Ficoll-Hypaque density gradient centrifugation isused to separate PBMC from red blood cells and neutrophils according toestablished procedures. Cells are washed with modified AIM-V (whichconsists of AIM-V (GIBCO) with 2 mM glutamine, 10 μg/ml gentamicinsulfate, 50 μg/ml streptomycin) supplemented with 1% fetal bovine serum(FBS). T cells are enriched by negative or positive selection withappropriate monoclonal antibodies coupled to columns or magnetic beadsaccording to standard techniques. An aliquot of cells is analyzed forcell surface phenotype including CD4, CD8, CD3 and CD14. For the purposeof illustration only, cells are washed and resuspended at aconcentration of about 5×10⁵ cells per ml of AIM-V modified as above andcontaining 5% FBS and 100 U/ml recombinant IL-2 (rIL-2) (supplementedAIM-V). Where the cells are isolated from and HIV⁺ patient, 25 nMCD4-PE40 (a recombinant protein consisting of the HIV-1-binding CD4domain linked to the translocation and ADP-ribosylation domains ofPseudomonas aeruginosa exotoxin A), or other similar recombinantcytotoxic molecule which selectively hybridizes to HIV is added to thecell cultures for the remainder of the cell expansion to selectivelyremove HIV infected cells from the culture. CD4-PE40 has been shown toinhibit p24 production in HIV-infected cell cultures and to selectivelykill HIV-1-infected cells.

To stimulate proliferation, OKT3 monoclonal antibody (Ortho Diagnostics)can be added to a concentration of 10 ng/ml and the cells are plated in24 well plates with 0.5 ml per well. The cells are cultured at atemperature of about 37° c in a humidified incubator with 5% CO₂ for 48hours. Media is aspirated from the cells and 1 ml of vector-containingsupernatant (described below) supplemented with 5 μl/m1 of protaminesulfate, 100 U/ml rIL-2, 100 U/m1 penicillin, 0.25 μg/m1 amphotericinB/ml and an additional 100 μg/m1 streptomycin (25 nM CD4-PE40 can beadded).

Cell Isolation and Characterization

In another aspect, cell surface markers can be used to isolate the cellsnecessary to practice the method of this invention. For example, humanstem cells typically express CD34 antigen while DCs express MHCmolecules and costimulatory molecules (e.g., B7-1 and B7-2), a lack ofmarkers specific for granulocytes, NK cells, B cells, and T cells. Theexpression of surface markers facilitates identification andpurification of these cells. These methods of identification andisolation include FACS, column chromatography, panning with magneticbeads, western blots, radiography, electrophoresis, capillaryelectrophoresis, high performance liquid chromatography (HPLC), thinlayer chromatography (TLC), hyperdiffusion chromatography, and the like,and various immunological methods such as fluid or gel precipitinreactions, immunodiffusion (single or double), immunoelectrophoresis,radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs),immunofluorescent assays, and the like. For a review of immunologicaland immunoassay procedures in general, see Stites and Terr (eds.) 1991Basic and Clinical Immunology (7th ed.) and Paul supra. For a discussionof how to make antibodies to selected antigens see Harlow and Lane(1989) supra.

Cell isolation or immunoassays for detection of cells during cellpurification can be performed in any of several configurations, e.g.,those reviewed in Maggio (ed.) (1980) Enzyme Immunoassay CRC Press, BocaRaton, Fla.; Tijan (1985) “Practice and Theory of Enzyme Immunoassays,”Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers B.V., Amsterdam; Harlow and Lane, supra; Chan (ed.)(1987) Immunoassay: A Practical Guide Academic Press, Orlando, Fla.;Price and Newman (eds.) (1991) Principles and Practice of ImmunoassaysStockton Press, NY; and Ngo (ed.) (1988) Non-isotopic ImmunoassaysPlenum Press, NY.

Cells can be isolated and characterized by flow cytometry methods such aFACS analysis. A wide variety of flow-cytometry methods are known. For ageneral overview of fluorescence activated flow cytometry see, forexample, Abbas et al. (1991) Cellular and Molecular immunology W.B.Saunders Company, particularly chapter 3, and Kuby (1992) ImmunologyW.H. Freeman and Company, particularly chapter 6. FACS machines areavailable, e.g., from Becton Dickinson.

Labeling agents which can be used to label cell antigen include, but arenot limited to monoclonal antibodies, polyclonal antibodies, proteins,or other polymers such as affinity matrices, carbohydrates or lipids.Detection proceeds by any known method, such as immunoblotting, westernblot analysis, tracking of radioactive or bioluminescent markers,capillary electrophoresis, or other methods which track a molecule basedupon size, charge or affinity.

Antibodies

Certain aspects of this method require the use of antibodies. Suchantibodies can be monoclonal or polyclonal. They can be antibodyderivatives or antibody variants. They can be chimeric, humanized, ortotally human. Using a protein or a polypeptide one of skill in the artcan generate additionally antibodies which specifically bind to thereceptor. A functional fragment or derivative of an antibody also can beused including Fab, Fab', Fab2, Fab′2, and single chain variableregions. Antibodies can be produced in cell culture, in phage, or invarious animals, including but not limited to cows, rabbits, goats,mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys,chimpanzees, apes, etc. So long as the fragment or derivative retainsspecificity of binding for the protein or fragment thereof it can beused. Antibodies can be tested for specificity of binding by comparingbinding to appropriate antigen to binding to irrelevant antigen orantigen mixture under a given set of conditions. If the antibody bindsto the appropriate antigen at least 2, 5, 7, and preferably 10 timesmore than to irrelevant antigen or antigen mixture then it is consideredto be specific.

Techniques for making such partially to fully human antibodies are knownin the art and any such techniques can be used. According to oneembodiment, fully human antibody sequences are made in a transgenicmouse which has been engineered to express human heavy and light chainantibody genes. Multiple strains of such transgenic mice have been madewhich can produce different classes of antibodies. B cells fromtransgenic mice which are producing a desirable antibody can be fused tomake hybridoma cell lines for continuous production of the desiredantibody. See for example, Russel et al. (2000) Infection and ImmunityApril 2000: 1820-1826; Gallo et al. (2000) European J. of Immun30:534-540; Green (1999) J. of Immun Methods 231:11-23; Yang et al.(1999A) J. of Leukocyte Biology 66:401-410; Yang (1999B) Cancer Research59(6): 1236-1243; Jakobovits (1998) Advanced Drug Delivery Reviews31:33-42; Green and Jakobovits (1998) Exp. Med. 188(3): 483-495;Jakobovits (1998) Exp. Opin. Invest. Drugs 7(4): 607-614; Tsuda et al.(1997) Genomics 42:413-421; Sherman-Gold (1997). Genetic EngineeringNews 17:14; Mendez et al. (1997) Nature Genetics 15:146-156; Jakobovits(1996) WEIR'S HANDBOOK OF EXPERIMENTAL IMMUNOLOGY, THE INTEGRATED IMMUNESYSTEM VOL. IV, 194.1-194.7; Jakobovits (1995) Current Opinion inBiotechnology 6:561-566; Mendez et al. (1995) Genomics 26:294-307;Jakobovits (1994) Current Biology 4:761-763; Arbones et al. (1994)Immunity 1:247-260; Jakobovits (1993) Nature 362:255-258; Jakobovits etal. (1993) Proc. Natl. Acad. Sci. USA 90:2551-2555; Kucherlapati, et al.U.S. Pat. No. 6,075,181.

Antibodies can also be made using phage display techniques. Suchtechniques can be used to isolate an initial antibody or to generatevariants with altered specificity or avidity characteristics. Singlechain Fv can also be used as is convenient. They can be made fromvaccinated transgenic mice, if desired.

The antibodies of this invention also can be modified to create chimericantibodies. Chimeric antibodies are those in which the various domainsof the antibodies' heavy and light chains are coded for by DNA from morethan one species. See, e.g., U.S. Pat. No.: 4,816,567.

The term “antibody variant” also includes “diabodies” which are smallantibody fragments with two antigen-binding sites, wherein fragmentscomprise a heavy chain variable domain (VH) connected to a light chainvariable domain (VL) in the same polypeptide chain (VH VL). See forexample, EP 404,097; WO 93/11161; and Hollinger et al., (1993) Proc.Natl. Acad. Sci. USA 90:6444-6448. By using a linker that is too shortto allow pairing between the two domains on the same chain, the domainsare forced to pair with the complementary domains of another chain andcreate two antigen-binding sites. See also, U.S. Pat. No. 6,632,926 toChen et al. which discloses antibody variants that have one or moreamino acids inserted into a hypervariable region of the parent antibodyand a binding affinity for a target antigen which is at least about twofold stronger than the binding affinity of the parent antibody for theantigen. The term also includes post-translational modification tolinear polypeptide sequence of the antibody or fragment. The term“antibody variant” further includes “linear antibodies”. The procedurefor making such variants is known in the art and described in Zapata etal. (1995) Protein Eng. 8(10): 1057-1062. Briefly, these antibodiescomprise a pair of tandem Fd segments (VH—CH 1-VH -CH1) which form apair of antigen binding regions. Linear antibodies can be bispecific ormonospecific.

Methods to Detect Nucleic Acids

Various methods are known for quantifying the expression of a gene ofinterest (e.g. CD40L and/or IL-12p35) and include but are not limited tohybridization assays (Northern blot analysis) and PCR basedhybridization assays. In assaying for an alteration in mRNA level suchas IL-12 p35 mRNA or CD40L mRNA, the nucleic acid contained in a samplecan be first extracted. For instance, mRNA can be isolated using variouslytic enzymes or chemical solutions according to the procedures setforth in Sambrook et al. (1989), supra or extracted by commerciallyavailable nucleic-acid-binding resins following the accompanyinginstructions provided by the manufacturers. The mRNA contained in theextracted nucleic acid sample can then detected by hybridization (e.g.,Northern blot analysis) and/or amplification procedures using nucleicacid probes and/or primers, respectively, according to standardprocedures.

Nucleic acid molecules having at least 10 nucleotides and exhibitingsequence complementarity or homology to the nucleic acid to be detectedcan be used as hybridization probes or primers in the diagnosticmethods. It is known in the art that a “perfectly matched” probe is notneeded for a specific hybridization. Minor changes in probe sequenceachieved by substitution, deletion or insertion of a small number ofbases do not affect the hybridization specificity. In general, as muchas 20% base-pair mismatch (when optimally aligned) can be tolerated. Forexample, a probe useful for detecting CD40L mRNA is at least about 80%identical to the homologous region of comparable size contained in apreviously identified sequence, e.g., see SEQ ID NOS: 1 or 3.Alternatively, the probe is at least 85% or even at least 90% identicalto the corresponding gene sequence after alignment of the homologousregion. The total size of fragment, as well as the size of thecomplementary stretches, will depend on the intended use or applicationof the particular nucleic acid segment. Smaller fragments of the genewill generally find use in hybridization embodiments, wherein the lengthof the complementary region may be varied, such as between about 10 andabout 100 nucleotides, or even full length according to thecomplementary sequences one wishes to detect.

Nucleotide probes having complementary sequences over stretches greaterthan about 10 nucleotides in length will increase stability andselectivity of the hybrid, and thereby improving the specificity ofparticular hybrid molecules obtained. One can design nucleic acidmolecules having gene-complementary stretches of more than about 25 andeven more preferably more than about 50 nucleotides in length, or evenlonger where desired. Such fragments may be readily prepared by, forexample, directly synthesizing the fragment by chemical means, byapplication of nucleic acid reproduction technology, such as the PCRTMtechnology with two priming oligonucleotides as described in U.S. Pat.No. 4,603,102 or by introducing selected sequences into recombinantvectors for recombinant production.

In certain embodiments, it will be advantageous to employ nucleic acidsequences of the present invention in combination with an appropriatemeans, such as a label, for detecting hybridization and thereforecomplementary sequences. A wide variety of appropriate indicator meansare known in the art, including fluorescent, radioactive, enzymatic orother ligands, such as avidin/biotin, which are capable of giving adetectable signal. A fluorescent label or an enzyme tag, such as urease,alkaline phosphatase or peroxidase, instead of radioactive or otherenvironmental undesirable reagents can also be used. In the case ofenzyme tags, colorimetric indicator substrates are known which can beemployed to provide a means visible to the human eye orspectrophotometrically, to identify specific hybridization withcomplementary nucleic acid-containing samples.

Hybridization reactions can be performed under conditions of different“stringency”. Relevant conditions include temperature, ionic strength,time of incubation, the presence of additional solutes in the reactionmixture such as formamide, and the washing procedure. Higher stringencyconditions are those conditions, such as higher temperature and lowersodium ion concentration, which require higher minimum complementaritybetween hybridizing elements for a stable hybridization complex to form.Conditions that increase the stringency of a hybridization reaction arewidely known and published in the art. See, Sambrook, et al. (1989)supra. One can also utilize detect and quantify mRNA level or itsexpression using quantitative PCR or high throughput analysis such asSerial Analysis of Gene Expression (SAGE) as described in Velculescu etal. (1995) Science 270:484-487. Briefly, the method comprises isolatingmultiple mRNAs from cell or tissue samples suspected of containing thetranscript. Optionally, the gene transcripts can be converted to cDNA. Asampling of the gene transcripts are subjected to sequence-specificanalysis and quantified. These gene transcript sequence abundances arecompared against reference database sequence abundances including normaldata sets for diseased and healthy patients. The patient has thedisease(s) with which the patient's data set most closely correlates andfor this application, includes the differential of the transcript.

In certain aspects, it may be necessary to use polynucleotides asnucleotide probes or primers for the amplification and detection ofgenes or gene transcripts. A primer useful for detecting differentiallyexpressed mRNA is at least about 80% identical to the homologous regionof comparable size of a gene or polynucleotide. For the purpose of thisinvention, amplification means any method employing a primer-dependentpolymerase capable of replicating a target sequence with reasonablefidelity. Amplification may be carried out by natural or recombinantDNA-polymerases such as T7 DNA polymerase, Klenow fragment of E. coliDNA polymerase, and reverse transcriptase.

General procedures for PCR are taught in MacPherson et al., PCR: APRACTICAL APPROACH, (IRL Press at Oxford University Press (1991)).However, PCR conditions used for each application reaction areempirically determined A number of parameters influence the success of areaction. Among them are annealing temperature and time, extension time,Mg²⁺ ATP concentration, pH, and the relative concentration of primers,templates, and deoxyribonucleotides.

After amplification, the resulting DNA fragments can be detected byagarose gel electrophoresis followed by visualization with ethidiumbromide staining and ultraviolet illumination. A specific amplificationof differentially expressed genes of interest can be verified bydemonstrating that the amplified DNA fragment has the predicted size,exhibits the predicated restriction digestion pattern, and/or hybridizesto the correct cloned DNA sequence. Other methods for detecting geneexpression are known to those skilled in the art. See, for example,.International PCI Application No. WO 97/10365, U.S. Pat. Nos. 5,405,783,5,412,087 and 5,445,934, 5,405,783; 5,412,087; 5,445,934;5,578,832;5,631,734; and LABORATORY TECHNIQUES IN BIOCHEMISTRY AND MOLECULARBIOLOGY, Vol. 24: Hybridization with Nucleic Acid Probes, Tijssen, ed.Elsevier, N.Y. (1993).

Methods for Detecting and Quantifying Protein or Polypeptides

A variety of techniques are available in the art for protein analysisand include, but are not limited to radioimmunoassays, ELISA (enzymelinked immunoradiometric assays), “sandwich” immunoassays,immunoradiometric assays, in situ immunoassays (using e.g., colloidalgold, enzyme or radioisotope labels), western blot analysis,immunoprecipitation assays, immunofluorescent assays and PAGE-SDS.

Ex Vivo Therapy

As noted above, this invention also provides ex vivo therapeutic methodsusing the dendritic cells or educated T cells produced by the methods ofthis invention. For example, dendritic cells are transformed with animmunogen can be used to activate cytotoxic and helper T cells in vitro.Alternatively, the transformed dendritic cells are introduced into amammal to activate the T cells in vivo. Yet further, T cells educated invitro can be introduced into a mammal where they are cytotoxic againsttarget cells bearing antigenic peptides corresponding to those the Tcells are activated to recognize on class I MHC molecules. These targetcells are typically cancer cells, or infected cells which express uniqueantigenic peptides on their MHC class I surfaces.

Similarly, helper T-cells, which recognize antigenic peptides in thecontext of MHC class II, can also be stimulated by the DCs of theinvention, which comprise antigenic peptides both in the context ofclass I and class II MHC. Helper T-cells also stimulate an immuneresponse against a target cell. As with cytotoxic Tcells, helper T-cellsare stimulated with the recombinant DCs in vitro or in vivo.

The dendritic cells and T cells can be isolated from the mammal intowhich the DCs and/or activated T cells are to admnistered.Alternatively, the cells can be allogeneic provided from a donor orstored in a cell bank (e.g., a blood bank).

In Vivo Therapy

T cells or dendritic cells produced by the methods of this invention canbe administered directly to the subject to produce T cells activeagainst a selected immunogen. Administration can be by methods known inthe art to successfully deliver a cell into ultimate contact with asubject's blood or tissue cells.

The cells are administered in any suitable manner, often withpharmaceutically acceptable carriers. Suitable methods of administeringcells in the context of the present invention to a subject areavailable, and, although more than one route can be used to administer aparticular cell composition, a particular route can often provide a moreimmediate and more effective reaction than another route. Preferredroutes of administration include, but are not limited to intradermal andintravenous administration.

Pharmaceutically acceptable carriers are determined in part by theparticular composition being administered, as well as by the particularmethod used to administer the composition. Accordingly, there is a widevariety of suitable formulations of pharmaceutical compositions of thepresent invention. Most typically, quality controls (microbiology,clonogenic assays, viability tests), are performed and the cells arereinfused back to the subject, preceded by the administration ofdiphenhydramine and hydrocortisone. See, for example, Korbling et al.(1986) Blood 67:529-532 and Haas et al. (1990) Exp. Hematol. 18:94-98.

Formulations suitable for parenteral administration, such as, forexample, by intraarticular (in the joints), intravenous, intramuscular,intradermal, intraperitoneal, intranodal and subcutaneous routes, andcarriers include aqueous isotonic sterile injection solutions, which cancontain antioxidants, buffers, bacteriostats, and solutes that renderthe formulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.Intraderaml and intravenous administration are the preferred method ofadministration for DCs or T cells of the invention.

The dose of cells (e.g., activated T cells, or dendritic cells)administered to a subject is in an effective amount, effective toachieve the desired beneficial therapeutic response in the subject overtime, or to inhibit growth of cancer cells, or to inhibit infection.

For the purpose of illustration only, the method can be practiced byobtaining and saving blood samples from the subject prior to infusionfor subsequent analysis and comparison. Generally at least about 10⁴to10⁶ and typically, between 1×10⁸ and 1×10¹⁰ cells are infusedintravenously or intraperitoneally into a 70 kg patient over roughly60-120 minutes. In one aspect, administration is by intravenousinfusion. Vital signs and oxygen saturation by pulse oximetry areclosely monitored. Blood samples are obtained 5 minutes and 1 hourfollowing infusion and saved for analysis. Cell re-infusions arerepeated roughly every month for a total of 10-12 treatments in a oneyear period. After the first treatment, infusions can be performed on anoutpatient basis at the discretion of the clinician. If the re-infusionis given as an outpatient, the participant is monitored for at least 4hours following the therapy.

For administration, cells of the present invention can be administeredat a rate determined by the effective donse, the LD-50 (or other measureof toxicity) of the cell type, and the side-effects of the cell type atvarious concentrations, as applied to the mass and overall health of thesubject. Administration can be accomplished via single or divided doses.The cells of this invention can supplement other treatments for acondition by known conventional therapy, including cytotoxic agents,nucleotide analogues and biologic response modifiers. Similarly,biological response modifiers are optionally added for treatment by theDCs or activated T cells of the invention. For example, the cells areoptionally administered with an adjuvant, or cytokine such as GM-CSF,IL-12 or IL-2.

In Vitro Assays and Kits

The present invention provides commercially valuable kits to practicethe maturation methods of the invention. In one aspect, the kitcomprises IFN' polypeptide, or an expression cassette for expressingIFN' mRNA in vivo or in vitro, and CD40L polypeptide, or an expressioncassette for expressing CD40L mRNA in vivo or in vitro expression ofCD40L. In another aspect, the kit comprises TNFa polypeptide, or anexpression cassette for expressing TNFa mRNA in vivo or in vitro, andCD40L polypeptide, or an expression cassette for expressing CD40L mRNAin vivo or in vitro expression of CD40L. The kits may further comprise aRNA polymerase for in vitro transcription.

Methods to Assess Immunogenicity

The immunogenicity of the antigen presenting cells or educated T cellsproduced by the methods of the invention can be determined by well knownmethodologies including, but not limited to the following:

⁵¹Cr-release lysis assay. Lysis of peptide-pulsed ⁵¹Cr-labeled targetsby antigen-specific T cells can be compared. “More active” compositionswill show greater lysis of targets as a function of time. The kineticsof lysis as well as overall target lysis at a fixed timepoint (e.g., 4hours) may be used to evaluate performance. Ware et al. (1983) 3.Immunol. 131:1312.Cytokine-release assay. Analysis of the types and quantities ofcytokines secreted by T cells upon contacting modified APCs can be ameasure of functional activity. Cytokines can be measured by ELISA orELISPOT assays to determine the rate and total amount of cytokineproduction. Fujihashi et al. (1993) J. Immunol. Meth. 160:181; Tanquayand Killion (1994) Lymphokine Cytokine Res. 13:259.In vitro T cell education. The compositions of the invention can beassayed for the ability to elicit reactive T cell populations fromnormal donor or patient-derived PBMC. In this system, elicited T cellscan be tested for lytic activity, cytokine-release, polyclonality, andcross-reactivity to the antigenic epitope. Parkhurst et al. (1996)Immunol. 157:2539.Transgenic animal models Immunogenicity can be assessed in vivo byvaccinating HLA transgenic mice with the compositions of the inventionand determining the nature and magnitude of the induced immune response.Alternatively, the hu-PBL-SCID mouse model allows reconstitution of ahuman immune system in a mouse by adoptive transfer of human PBL. Theseanimals may be vaccinated with the compositions and analyzed for immuneresponse as previously mentioned in Shirai et al. (1995) J. Immunol.154:2733; Mosier et al. (1993) Proc. Natl. Acad. Sci. USA 90:2443.Proliferation Assays. T cells will proliferate in response to reactivecompositions. Proliferation can be monitored quantitatively bymeasuring, for example, ³H-thymidine uptake. Caruso et al. (1997)Cytometry 27:71.Primate models. A non-human primate (chimpanzee) model system can beutilized to monitor in vivo immunogenicities of HLA-restricted ligands.It has been demonstrated that chimpanzees share overlapping MHC-ligandspecificities with human MHC molecules thus allowing one to testHLA-restricted ligands for relative in vivo immunogenicity. Bertoni etal. (1998) Immunol. 161:4447.Monitoring TCR Signal Transduction Events. Several intracellular signaltransduction events (e.g., phosphorylation) are associated withsuccessful TCR engagement by MHC-ligand complexes. The qualitative andquantitative analysis of these events have been correlated with therelative abilities of compositions to activate effector cells throughTCR engagement. Salazar et al. (2000) Tnt. J. Cancer 85:829; Isakov etal. (1995) J. Exp. Med. 181:375).

In accordance with the above description, the following examples areintended to illustrate, but not limit, the various aspects of thisinvention.

EXPERIMENTAL EXAMPLES Reagents

Histopaque 1077 and Tween 20 were purchased from Sigma (St Louis, Mo.).PBS and X-VIVO 15 were purchased from Cambrex (East Rutherford, Ni).AIM-V medium, Iscove's modified Dulbecco's medium and RPMI 1640 mediumalong with Trypan Blue and Fetal Bovine Serum (FBS) were purchased fromInvitrogen (Carlsbad, Calif.). Viaspan was purchased from Dupont PharmaLabs (Wilmington, Del.). GM-CSF, IL-4, TNF-α, IL-1β, IL-6 and IFN-γ wereall purchased from R&D Sytems (Minneapolis, Minn.). PGE₂ was purchasedfrom Cayman Chemicals (Ann Arbor, Calif.). Soluble CD40L was purchasedfrom Alexis Biochemicals (San Diego Calif.). Human AB serum waspurchased from Valley Biochemical (Winchester, Va.).

Chemokines CCL19 and CCL21 were purchased from Peprotech (Rocky Hill,N.J.). Phenotyping antibodies (HLA-ABC, HLA-DR, CD80, CD86, CD83, CD14,and negative isotype controls), ELISpot antibody pairs (IFN-γ and IL-2)ELISA sets (IL-12 and IL-10) and streptavidin-HRP were all purchasedfrom BD Pharmingen (San Diego, Calif.) along with BD Opt EIA reagent setB pH9.5. AEC peroxidase substrate was purchased from Vector labs (VectorLabs, Burlingame, Calif.). Blocking anti-CD40L antibody was purchasedfrom eBioscience. CD1d/α-galactosylceramide (KRN7000) tetramer andnative KRN7000 were kind gifts from Kirin Brewery, PharamaceuticalsDivision, Tokyo, Japan. MART-1/HLA-A201 tetramers were purchased fromBeckman-Coulter (Miami, Fla.)

DC Generation

Human PBMCs were isolated from Leukapheresis collections from healthyvolunteers provided by Life Blood (Memphis, Tenn.). PBMCs were preparedby Ficoll-Histopaque density centrifugation and washed four times in PBSat room temperature. 2×10⁸ PBMCs were re-suspended in 30 ml AIM-V mediumand allowed to adhere to 150 cm³ plastic flasks for 2 hours at 37° C.Non-adherent cells were removed and remaining cells cultured in X-VIVO15 medium, supplemented with GM-CSF (1000 U/ml) and IL-4 (1000 U/ml),for 5-6 days at 37°′C, 5% CO₂.

Cloning of CD40L

T cells were stimulated with PMA in RPMI for 1 hr. Cells were harvestedand washed with PBS once. Total RNA was extracted using QIAGEN RNeasyprocedure. One microgram of total RNA from activated T cells was takeninto one tube RT-PCR reaction using Gene Amp Gold kit (AppliedBioscience) using a high fidelity Advantage Polymerase (Clontech). Genespecific primers for CD40L sequence correspond to bases 47 and 859 ofCD40L sequence CD40L 5′ primer: 5′-GCATCATCGAAACATACAACC-3′ (SEQ ID NO.11) and CD40 3′ primer: 5′-GTATTATGAAGACTCCCAGCG-3′ (SEQ ID NO. 12). ThePCR fragment was purified and subcloned into pCR2.1 vector using T4 DNAligase (Invitrogen). Sequence analysis of the CD40L open reading frameand alignment with a GenBank consensus sequence revealed presence of twomutations. One mutation was conservative and did not lead to amino acidchange. Another substitution resulted in a functional amino acid changeAsn-Ser. Site directed mutagenesis was performed to correct thenon-conservative amino acid change back to asparagine. Briefly, 10-40 ngof CD40L PCR2.1 plasmid DNA was used in site directed mutagenesis usingcustom 5′ phosphorylated and HPLC purified primers (QIAGEN), PFU Ultraenzyme with accompanying 10X PCR buffer (Stratagene) and dNTPS(Clontech) Following the PCR reaction, Dpn I restriction enzyme(Promega) was added and incubated for 1 hour at 37° C. to digest awayparental template. Five microliters of this reaction was thentransformed into Oneshot MACH T1R competent cells (Invitrogen) andplated out on freshly made ampicillin containing LB plates. Six colonieswere selected and grown as 3 mL cultures overnight in LB containingampicillin. DNA was isolated using plasmid miniprep (QIAGEN). An aliquotof purified DNA for each clone was submitted to the University of NorthCarolina (UNC) sequencing facility for sequence analysis of the CD40Lopen reading frame using M13F and M13R primers (Invitrogen). All theclones were then aligned to a consensus GenBank Sequence for CD40L usingDNASTAR Seqman analysis software. Clone #2 (renamed CD40L WT PCR 2.1)was selected for containing the correct mutagenized bases.

Generation of mRNAs for Transfection of DCs

CD40L WT PCR 2.1 plasmid was linearized using Spcl restriction enzymeand purified by phenol/chloroform extraction followed by ethanolprecipitation. The linear template was reconstituted in water andtranscribed in vitro using mMessage mMachine T7 Ultra kits (Ambion)following the manufacturer's directions. An aliquot of RNA was saved forfinal analysis prior to proceeding to polyadenylation reaction.Polyadenylated RNA was purified using RNeasy column (QIAGEN) followingprotocol for RNA cleanup. RNA was eluted in water and stored inindividual size aliquots below −150° C. PolyA tail length was determinedby the comparative analysis of non-polyadenylated RNA and final productusing RNA Bioanalyzer 2100.

Electroporation of DCs

Prior to electroporation, DCs were harvested and washed in PBS and thenre-suspended in chilled Viaspan® (Barr Laboratories) at 4×10⁷/ml in0.5ml or 2.5×10⁷/ml in 0.2 ml and placed on ice. DCs were mixed withmRNA (1 or 2 μg/10⁶ for mRNA encoding antigen and 4 μg/10⁶ for CD40LmRNA) and placed in a 4 mm gap electroporation cuvette andelectroporated using Biorad apparatus Immediately after electroporation,DCs were washed in X-VIVO 15 medium and finally re-suspended in X-VIVO15 supplemented with GM-CSF (800 U/ml) and IL-4 (500 U/ml) at 1×10⁶/mland cultured for either 4 or 24 hours at 37° C. in low adherence sixwell plates (BD Biosciences, Franklin Lakes, N.J.). Additionalmaturation stimuli, described below, were also added at this point.

DC Maturation—Cytokine Cocktail Process

Immature DCs are optionally transfected with various antigen-encodingmRNAs and are then treated with a “cytokine cocktail” comprising ofTNF-α (10 ng/ml), IL-1β (10 ng/ml), IL-6 (100 ng/ml) and PGE₂ (1 μg/ml)and cultured in medium containing GM-CSF and IL-4 overnight at 37° C.

DC Maturation—CD40L Base Process.

Following electroporation, DCs transfected with CD40L mRNA were treatedwith IFN-γ (1000 U/ml) or TNF-α (10 ng/ml) or a combination of IFN-γ andPGE₂ (1 μg/ml). By comparison, immature DCs were transfected withvarious antigen-encoding mRNAs and were then treated with a “cytokinecocktail” comprising of TNF-α (10 ng/ml), IL-1β (10 ng/ml), IL-6 (100ng/ml) and PGE₂ (1 μg/ml) or soluble CD40L (200 ng/ml) plus enhancer (1μg/ml) with either simultaneous or sequential addition of 1000 U/mlIFN-γ.

DC Maturation—PME-CD40L Process.

Immature DCs were phenotypically matured on Day 5 of culture with TNF-α(10 ng/ml), IFN-γ (1000 U/ml) and PGE₂ (1 μg/ml). On day 6, DCs wereharvested and electroporated with antigen and CD40L mRNA as describedabove, and cultured in X-VIVO 15 media containing 800 U/ml GM-CSF and500 U/ml IL-4 for 4 hrs prior to harvest, or formulation for vaccineproduction.

DC Maturation with the CD40L Base Process, in Combination withα-Galactosylceramide (KRN7000)

100 ng/ml of KRN7000 was pulsed onto the CD40L base process DCsimmediately post electroporation in combination with 500 U/ml IFN-γ and1 μg/ml PGE₂, for 24 hrs of culture.

Flow Cytometry Analysis of DCs

10⁶ DCs were harvested and re-suspended in chilled PBS/1% FCS.Phycoerythrin (PE) or FITC conjugated antibodies specific for MHCmolecules (HLA-ABC, HLA-DR), co-stimulatory molecules (CD80, CD86),maturation markers (CD83) and monocyte markers (CD14) were mixed with1×10⁵ DCsper well in a 96 well plates (BD Biosciences) and incubated at4° C. for a minimum of 15 minutes. Isotype matched antibodies were usedas controls. After thorough washing, fluorescence analysis was performedwith a FACScalibur flow cytometer (BD Biosciences) using CellQuestsoftware (BD Biosciences).

Intracellular expression of CD40L was determined as follows: 2×10⁵ DCsor HeLa cells were harvested at various time points post transfectionwith CD40L mRNA and re-suspended in 250 μL of Cytofix/Cytoperm solution(BD Biosciences) for a minimum of 10 minutes up to 2 hours at 4° C.Cells were washed twice with 2 ml staining buffer (PBS, BSA, NaN₃, andEDTA), re-suspended in 0.5 ml staining buffer and stored over night at4° C. Cells were re-suspended in 2.0 ml Perm/Wash solution (BDBiosciences) for 15 minutes, centrifuged and re-suspended in 100 μlPerm/Wash solution. 20 μL of mouse anti-human CD40L PE and anti-humanCD40 APC (BD Biosciences) or mouse IgG1 PE and IgG1 APC (BD Biosciences)was added to each DC preparation collected and permeabilized at eachtime point, and incubated at 4° C. for 30 minutes in the dark. Cellswere washed twice with 1 ml Perm/Wash solution and re-suspended instaining buffer prior to flow cytometric analysis.

Intracellular cytokine staining (ICS) was performed as follows: 1×10⁶/mlprimed CD8+ T cells, removed from co-culture on day 19 and re-stimulatedin 200 μl R10 media with PME DC targets (RCC, survivin, G250, hTERT oreGFP) at 37° C.; 5% CO₂ for 1 hour prior to the addition of brefeldin A(BD GolgiPlug, Cat No. 555029) at 1 μl/ml. Cells incubated at 37° C. fora further 16 hours. Cells were washed and resuspended in 150 μl FACSbuffer with 5 μl CD8 per CP-cy5.5 (BD 341051) and incubated at 4° C.After 30 minutes cells were washed twice and resuspended in 2%paraformaldehyde (PFA). Cells were subsequently washed after 10 minutes,and then permeabilized in 0.1% saponin for 10 minutes at roomtemperature (RT), prior to incubation with 2 μl of blocking antibody,Mouse IgG1 pure (BD 349040). After 10 minutes incubation at RT, 0.5 μlIFN-γ -APC (BD 554681), 10 μl IL-2-FITC (BD 554702) and 10 μl CD69-PE(BD 555531) antibodies were added to each sample tube. Samples wereincubated for 30 minutes in the dark at RT. Cells were resuspended in 2%PFA following a final wash in 0.1% saponin. Analysis undertaken by FACScytometery, collecting 100,000 events.

CD40L Functional Analysis when Expressed from mRNA in HeLa Cells

HeLa cells were grown in 10% FBS/DMEM and then harvested andelectroporated in 4 mm cuvettes with GFP and CD40L RNA (20 μg each/5×10⁶cells). Post-transfection recovery was ˜70% and the cells were plated in6 well dishes and allowed to grow overnight. Following the overnightincubation, transfected HELA cells were harvested by scraping andstained with either mouse IgG1-PE or anti-human CD40L-PE (both from BDBiosciences, San Diego, Calif.) to look for cell surface expression ofCD40L. 2×10⁵ cells/tube were stained with 10 μg/ml of antibody in 1%FBS/PBS for 30 minutes at 4° C. The cells were analyzed using aFACScaliber flow cytometer and Cellquest software (BD Biosciences). Toanalyze the function of the HeLa expressed CD40L, 1×10⁶ immaturedendritic cells were co-cultured with 1×10⁶ HeLa cells in 5% huABserum/RPMI supplemented with 1000 U/ml of IFN-γ (R&D Systems,Minneapolis, Minn.) in 6 well dishes (2 mls total volume) overnight. Ablocking CD40L monoclonal antibody (24-31 from eBioscience) was includedat 10 μg/ml in matched wells to confirm that cell surface expression ofthe protein was required for stimulating the dendritic cells. Theculture supernatant was harvested after 18-24 hours and expression ofthe cytokines IL-10 and IL-12 analyzed by ELISA (BD Biosciences).

Migration Assay

Chemotaxis of DCs was measured by migration through a 8 μm pore sizepolycarbonate filter in 24 well transwell chambers (corning Costar,Acton, Mass.). 5% human AB serum in Iscoves modified Dulbecco's mediumor AIM-V medium containing 3-300 ng/ml CCL19, 5-250 ng/ml CCL21, acombination of both or medium alone was added to the lower chamber.1-5×10⁵ DCs in 0.1 ml were added to the upper chamber and incubated for2-3 hours at 37° C. Lower chamber harvested into 5 ml tubes (BDBiosciences) and re-suspended in 0.1 ml PBS and viable cell countsundertaken using trypan blue.

ELISpot

PVDF membrane ELISpot plates (Millipore, Ballerica, Mass.) were coatedwith 5 μg/mL monoclonal anti-IFN-γ or anti-IL-2 capture antibody (BDPharmingen, San Diego, Calif.) and incubated at 4° C. for 24 hours.After incubation, plates were washed with PBS/0.05% Tween 20, andblocked with 5% human AB serum/RPMI 1640 medium for 1 hour. PBMCs,T-cells, or CD8 enriched T cells, were plated at 1×10⁵ cells/well andmRNA transfected, antigen-loaded DC targets at 1×10⁴ cells/well for a10:1 effector:target ratio, and incubated at 37° C., 5% CO₂ for aminimum of 16 hours.

Following incubation, plates were washed 6 times, and anti-IFN-γdetection antibody (BD Pharmingen) or anti-IL-2 detection antibody (BDPharmingen) was added to the appropriate plates at 1 μg/ml for 2 hours.After a further six washes, Streptavidin-HRP (BD Pharmingen) was addedto each well for 1 hour. Finally, after another wash cycle, colordevelopment was undertaken with AEC Peroxidase Substrate for 5-15minutes and stopped with water. The plates were left to air dry prior toanalysis on CTL Immunospot Plate Reader (CTL, Cleveland, Ohio).

ELISA

The method as laid out by BD Pharmingen for IL-12 and IL-10 ELISA sets(BD Pharmingen) using BD Opt EIA reagent set B pH 9.5. Briefly, ELISAplates (BD Biosciences) were coated with anti-IL-12p70 or anti-IL-10ELISA capture antibody in coating buffer for 24 hours at 4° C. Platesunderwent blocking with 200 μl per well 10% FCS/PBS for one hour priorto the addition of standards (BD Pharmingen) and supernatant samples, induplicate, at 100 μl per well and incubated at room temperature for 2hours. Plates were washed and anti-cytokine detection antibody added,incubated for one hour, the plates washed and solutions replaced with100 μl of streptavidin-HRP and further incubated for one hour at roomtemperature. Again plates were washed and color development substratesapplied for 10-20 minutes, followed by cessation of color developmentwith stop solution. Plate analysis undertaken using Bio-Tek instrumentsELx800 plate reader with KC junior software (Winooski, Vt.). The resultsshow the number of picograms/ml/10⁶ DCs. Because the assays were set upso that 1 ml corresponds to 10⁶ DCs, the results can also be expressedas number of picograms/10⁶ DCs. For example, 3000 pg/ml/10 ⁶ DCs isequivalent to 3000 pg/10⁶ DCs.

CTL Induction

Mature dendritic cells transfected with mRNAs were co-cultured with CD8purified T-cells. All co-cultures were performed in R-10 media (10% FBS,RPMI-1640 supplemented with 10 mM HEPES pH 7.4, 1 mM sodium pyruvate,0.1 mM non-essential amino acids, 2 mM sodium glutamate, 55 μMβ-mercaptoethanol). All cell culture reagents were from Invitrogen(Carlsbad, Calif.). CD8⁺ cells were purified using the CD8⁺ T CellIsolation kit II (Miltenyi Biotec, Auburn, Calif.) from non-adherentcells harvested from the monocyte adherence step. The CD8⁺ cells aremixed with dendritic cells prepared as described above at 10:1 CD8⁺:DC.For the first seven days the cells were cultured in media supplementedwith 0.2 U/ml IL-2 (R&D Systems, Minneapolis, Minn.) and then aliquotedinto 24-well tissue culture dishes at 1 ml (1×10⁶ CD8⁺ cells)/well.Following this initial seven day incubation the CD8+ cells wereharvested, counted and re-cultured with fresh DC stimulators at 10:1 inmedia supplemented with 5 U/ml IL-2. Again the cells were cultured forone week and then restimulated with fresh DC and 20 U/ml IL-2. CTLassays were performed 3 or 7 days following the third stimulation.

CTL Assay

T2 cells (ATCC Number CRL-1992) were previously pulsed with 10 μg/ml ofeither the HLA-A201 restricted MART-APL peptide (LAGIGILTV; SEQ ID NO:24) or native peptide (AAGIGILTV; SEQ ID NO: 25) or PSA-1 peptide(FLTPKKLQCV; SEQ ID NO: 26) by overnight incubation in FBS/RPMI media,and washed prior to use as CTL targets. Dendritic cell targets weretransfected with either GFP mRNA, MART-1 APL mRNA, or Flu-M1 mRNA, asdescribed above and incubated overnight without maturation. Pulsed T2cells were incubated with 100 μCi of Na⁵¹Cr (Perkin-Elmer Life andAnalytical Sciences, Inc., Boston, Mass.) for 90 minutes at 37° C.Excess ⁵¹Cr was washed away and 5000 labeled targets incubated withvarious E:T ratios of CD8+cells for 4 hours. Non-specific lysis wasreduced by the addition of unpulsed T2 cells at 25,000 cells per well.Released ⁵¹Cr was measured in the supernatant by scintillation counting.Total release was calculated by addition of 1% Triton X-100 to thetargets while spontaneous release was calculated by addition of mediaalone. Percent lysis was calculated using the formula (sample cpmreleased-spontaneous cpm)/(total cpm released-spontaneous cpm released).

Induction of MART-1 Specific CTLs Employing KRN7000-pulsed CD40L BaseProcess Matured DC

DCs were generated as described above, employing the ‘CD40L baseprocess’, and loaded with mRNA encoding MART-1. Post electroporation,DCs were incubated with KRN7000, IFN-γ and PGE₂. DCs and PBMCs wereco-cultured at a 1:10 ratio in the presence of 20 U/ml IL-2. PBMCs wererestimulated three times under the same conditions, and the frequency ofCTL induction determined by staining with MART-1/A2 tetramers, and theexpansion of NKT-cells enumerated using KRN7000/CD1d tetramers by FACS.

RESULTS OF EXPERIMENTAL EXAMPLES

Sequential Maturation with Interferon-γ and CD40L Optimizes IL-12p70Secretion

Immature DCs were prepared by 6 day culture of adherent cells PBMCs inX-VIVO 15 media, inclusive of GM-CSF and IL-4. DCs were recovered on Day6 and electroporated with 2 μg of eGFP encoding mRNA per million DCs,and matured for 36 hrs with “cytokine cocktail”. Alternatively,maturation was achieved by culturing the DCs in the presence of IFN-γand soluble CD40L, applied simultaneously, or sequentially. DCs weremonitored for increased expression of co-stimulatory molecules, but mostimportantly for the secretion of IL-12p70 versus IL-10. FIG. 1 showsthat DCs matured with the cytokine cocktail secrete excess IL-10 incomparision to IL-12p70 into the culture supernatant over the 36 hrculture period. By contrast, DCs matured simultaneously with solubleCD40L plus IFN-γ secrete excess IL-12p70. However, sequentialapplication of IFN-γ for 18 hrs, followed by the addition of solubleCD40L directly to the culture, and an additional 18hr culture periodresulted in significantly enhanced levels of IL-12p70 secretion.Unexpectedly, the application of soluble CD40L, followed by IFN-γ,prevented significant secretion of IL-12p70. In conclusion, thesequential delivery of an innate stimulus to “prime” DC maturation(IFN-γ), followed by a surrogate T-helper cell signal delivered bysoluble CD40L, optimizes DC maturation for IL-12p70 secretion.

Co-Culture of HeLa Cells, Transfected with mRNA Encoding CD40L, withImmature DCS Results in the Induction of DC Derived IL-12p70.

FIG. 2 shows that HeLa cells transfected with mRNA encoding CD40Lresults in significant cell surface expression of CD40L protein after 24hrs of culture, as defined by an anti-CD40L antibody and flow cytometry.CD40L mRNA transfected HeLa cells were co-cultured with immature DCs, inthe presence of 1000 U/ml IFN-γ. Table II shows that HeLa cellstransfected with an extended poly-A tail (>100 ‘A's) are capable ofinducing significant IL-12p70 secretion when cultured with immature DCsover the 18 hr culture period. Importantly, the inclusion of a blockinganti-CD40L antibody prevents IL-12p70 secretion, and confirms theidentity and functional importance of protein encoded by the transfectedmRNA sequence.

TABLE II HeLa cells transfected with CD40L encoding mRNA, whenco-cultured with immature DCs in the presence of IFN-γ, results in thesecretion of IL-12p70. Inclusion of ‘blocking’ anti-CD40L antibody inthe culture prevents the induction of IL-12p70. “Cocktail” maturedImmature DCs and reactivated DCs IL-12p70 (pg/ml) IL-12p70 (pg/ml) DC'salone — 4.9 (a) HeLa/>100 polyA + 372 26.3 IFN-γ (b) HeLa/>100 polyA + —2.5 IFN-γ + 24-31 (a) HeLa cells were transfected with 4 μg of CD40LmRNA bearing greater than 100 nucleotide poly-A tail, and incubated withDCs in the presence of IFN-γ. (b) As (a), but in the presence ofblocking anti-CD40L antibody (24-31).Dendritic Cells Transfected with CD40L mRNA, and Cultured in thePresence of IFN-γ Secrete IL-12p70.

Immature DCs were harvested after 6 days in culture with GM-CSF andIL-4, and transfected with a titration of CD40L mRNA (>100-polyA), andimmediately cultured in the presence of 1000 U/ml IFN-γ. FIG. 3 showsthat supernatants harvested after 18 hrs of culture contain excessIL-12p70 over IL-10, and that at least 4 μg of CD40L mRNA per millionDCs is required for optimal cytokine secretion. Increasing the CD40LmRNA payload above 4 μg per million DCs results in a significantreduction in DC yield post maturation (data not shown). In a parallelexperiment, immature DCs were transfected with 4 μg CD40L mRNA permillion cells, and a titration of IFN-γ immediately applied to thecultures. FIG. 4 shows that at least 100 U/ml of IFN-γ is required tosupport optimal induction of IL-12p70. FIG. 5a shows that IL-12p70appears at detectable levels 6 to 8 hrs post transfection and coculturewith IFN-γ, with optimal accumulation in the culture supernatant beingrecorded between 20 and 24 hrs. By contrast, the substitution of 10ng/ml TNF-α for IFN-γ also supports IL-12 production, but at reducedlevels (FIG. 5b ). Moreover, IFN-γ results in concomitantly lower levelsof IL-10 production than does TNF-α. (FIG. 5c )

Induction of IL-12p70 by DCs Transfected with CD40L mRNA is Dependent on“Intracellular Signaling” as Opposed to Cell-Cell Interactions.

FIG. 2 demonstrates that CD40L protein translated from mRNA can beexpressed on the cell surface of the transfected cells, and that theprotein retains the ability to appropriately signal DCs for IL-12p70secretion as a consequence of its interaction with its counterpart onDCs, namely CD40. To determine the cellular distribution of CD40L intransfected DCs, and to confirm its functional identity, DCs wereharvested at various time points post transfection, the presence ofCD40L on the cell surface, or intracellular compartments was determinedFIGS. 6a and 6b show that the majority of CD40L is localized within anintracellular compartment, and that significant protein expression (27%DCs CD40L positive) was not apparent until 60 minutes post transfection.Thus, although IFN-γ is applied immediately post transfection, thedelivery of the maturation events is sequential, with the IFN-γ signalpreceding that of CD40L. As shown in FIG. 1, sequential maturation ofDCs with IFN-γ and CD40L optimizes for IL-12p70 secretion. In addition,FIG. 7 shows that CD40L transfected and IFN-γ treated DCs, when culturedin the presence of excess blocking anti-CD40L antibody for 18 hrs posttransfection, still secrete significant levels of IL-12p70. This datashows that CD40L/CD40 interactions, which are required for IL-12p70production in this system, can take place within the intracellularcompartment.

Frequency of CD40L Positive Cells Over Time

Immature DCs were transfected with 4 μg CD40L mRNA per 10⁶ DC, andco-matured with 1000 U/ml IFN-γ. Alternatively, and by way of negativecontrol for CD40L staining, immature DCs were matured with ‘cytokinecocktail’. Maximum frequency of expression is achieved around 3 to 4 hrspost transfection with CD40L RNA (see FIG. 6b ), although 80% of DCsexpress CD40L when the cells are fixed and permeabilized, cell surfacestaining only detects approximately 15% of the DCs (FIG. 6c ). This datashows that the bulk of the CD40L protein is retained within the DC, andis not expressed at the cell surface. CD40L protein is transientlyexpressed, with the majority of DCs becoming CD40L negative 26 hrs posttransfection. The expression of CD40, the cognate receptor molecule forCD40L, is not altered by transfection of DCs with mRNA encoding CD40L,when compared to DCs receiving ‘cytokine cocktail’ only.

PGE₂ is Required to Induce DC Migration on Maturation with CD40L andIFN-γ

In addition to the capacity to secrete IL-12p70 and exhibit a maturephenotype, typically defined as cells expressing elevated levels ofco-stimulatory molecules such as CD80, CD83 and CD86 (see Table III),DCs must display the capacity to migrate, if they are going to becapable of homing to a lymph node in vivo. Several studies have shownthat PGE₂ primes mature DCs for migration (Luft et al. (2002) Blood 100:1362, Scandella et al. (2002) Blood 100: 1354). FIG. 8 shows that theinclusion of 1 μg/ml PGE₂, in addition to IFN-γ, enables the maturingDCs to migrate, and that the acquisition of this migratory potential isproportional to the CD40L mRNA payload. Thus, CD40L contributes to notonly the maturing DC phenotype, and dominant IL-12p70 profile (see TableII), but also to priming for migration. By contrast, DCs matured bytransfection with CD40L mRNA and cultured in the presence of IFN-γ, butin the absence of PGE₂, fail to migrate (data not shown), despitedisplaying significant cell surface expression of the chemokinereceptor, CCR7.

TABLE III Phenotypic analysis and secreted cytokine profile of DCsundergoing maturation induced by either 'Cytokine Cocktail', or CD40Lplus IFN-γ and PGE₂ (d) Mart- (a) Flu/ (b) Mart- (c) Flu/ APL/ eGFP APLCD40L CD40L mRNA mRNA mRNA mRNA DC Immature Cytokine Cytokine IFN-g/IFN-g/ markers DC Cocktail Cocktail PGE₂ PGE₂ HLA-ABC 99.7% 98.6% 99.5%99.9% 99.9% HLA-DR 95.0% 99.6% 99.7% 99.8% 99.5% CD83 23.2% 98.3% 99.2%99.6% 99.3% CD14 0.3% 1.7% 2.9% 3.2% 4.9% CD56 2.8% 3.3% 3.2% 2.8% 2.1%CD19 1.8% 1.1% 2.1% 3.2% 3.2% CD3 2.8% 2.4% 3.1% 2.8% 3.1% CD86 59.3%99.7% 100.0% 100.0% 100.0% CD80 28.8% 99.0% 99.5% 99.2% 99.5% CD1a 51.6%49.1% 52.2% 48.6% 49.9% CD209 95.8% 95.5% 96.1% 96.4% 95.9% CCR7 3.2%47.4% 35.5% 35.4% 36.2% (e) IL-12 N/A 27.5 59.0 1456.3 1350.0 (pg/ml)(f) IL-10 N/A 948.8 810.0 187.7 165.5 (pg/ml)

DCs were prepared from adherent monocytes and cultured for 6 days inGM-CSF/IL-4. On harvesting, DCs were transfected with various mRNApayloads and subjected to maturation for a further 24 hrs. DCs wereagain harvested, and the cells stained for various cell surface markers,particularly those associated with increased function, namelyco-stimulation and migration. Supernatants from the maturation cultureswere collected and subjected to IL-12p70 and IL-10 cytokine analysis.

-   (a) DCs were transfected with 2 μg per million cells with flu mRNA    as antigen-encoding payload, in addition to 4 μg per million cells    eGFP mRNA. eGFP mRNA allows for confirmation of transfection by    FACS, and to act as a substitute control for the 4μg per million    cells CD40L mRNA maturation payload, in the alternate process. These    flu/eGFP transfected DCs were matured in the presence of the    “cytokine cocktail”.-   (b) DCs were transfected with 2 μg per million cells with MART-APL    mRNA as antigen-encoding payload, and subjected to maturation with    the “cytokine cocktail”.-   (c) DCs were transfected with 2 μg per million cells with flu mRNA    as antigen-encoding payload, concomitant with 4 μg per million cells    CD40L mRNA as the maturation payload. These cells were immediately    placed in culture with IFN-γ and PGE₂ as described in materials and    methods.-   (d) DCs were transfected with 2 μg per million cells with MART-APL    as antigen-encoding payload, concomitant with 4 μg per million cells    CD40L mRNA as the maturation payload. These cells were immediately    placed in culture with IFN-γ and PGE₂ as described in materials and    methods.-   (e) IL-12p70 secretion from DCs undergoing maturation.-   (f) IL-10 secretion from DCs undergoing maturation.    DCs Sequentially Matured via Transfection with CD40L mRNA and    IFN-γ/PGE₂ Invoke Potent T-Cell Recall Responses.

To determine the “immunopotency” of DCs matured via CD40L mRNAtransfection and IFN-γ/PGE₂, DCs were co-transfected with 2 μg mRNAencoding flu matrix protein per million DCs in addition to the CD40LmRNA and IFN-γ/PGE₂ culture environment. 18 hrs post transfection, DCswere harvested, washed, and co-cultured with autologous T-cells in IFN-γELISpot assays. FIG. 9 shows that DCs matured via CD40L/IFN-γ/PGE₂display increased immunopotency, compared to DCs transfected with flumRNA and matured with ‘cytokine cocktail’, as defined by the frequencyof flu-specific IFN-γ spots in the assay.

DCs Sequentially Matured via Transfection with CD40L mRNA and IFN-γ/PGE₂Invoke Primary Responses.

Recall responses, such as that described in FIG. 9, are less dependenton the presence of DCs expressing optimized co-stimulatory molecules andsupporting cytokine environments. Therefore, DCs were tested for theirability to invoke primary immune responses to the melanoma associateantigen, MART-1, to which many healthy donors maintain a high naiveT-cell precursor frequency. As HLA-A201 donors were preferentially used,DCs were transfected with an mRNA encoding MART-1 in which the A2restricted determinant was optimized by mutation of the mRNA sequence bysite directed mutagenesis, such that the alanine at position 27 wassubstituted by leucine, and here referred to as MART-APL (Valmori, D etal (1998) J. Immunol. 160:1750). DCs co-transfected with 2 μg MART-APLmRNA with 4 μg CD40L mRNA and immediately pulsed with IFN-γ/PGE₂ for 18hrs were compared to DCs loaded solely with MART-APL, and maturedovernight with the “cytokine cocktail”. Antigen-loaded and matured DCswere added to purified autologous CD8¹ T-cells, and cultured for 7 daysin the presence of 0.2 U/ml human IL-2. After this period, T-cells wererecovered and co-cultured with a second round of antigen-loaded DCstimulators as appropriate in an IL-2 ELISpot assay. FIG. 10 shows thatCD8+ T-cells cultured in the presence of DCs matured via CD40L andIFN-γ/PGE₂ results in a highly significant increase in T-cells capableof IL-2 secretion in a specific response to the optimized MART-APLepitope originally encoded within the MART-APL mRNA sequence. Inconclusion, DCs exposed to sequential maturation via IFN-γ/PGE₂ andCD40L are significantly more potent at raising primary immune responsesthan DCs matured with the currently accepted standard “cytokinecocktail”. Moreover, FIG. 11 shows that CTLs generated with MART-APLloaded DCs matured with the ‘cytokine cocktail’ fail to mediateCD4-independent CD8-mediated cytotoxicity against T2 cells pulsed withthe appropriate HLA-A2 restricted MART-APL peptide (FIG. 11b ). Bycontrast, CTLs generated on CD40L/IFN-γ/PGE₂ matured DCs are fullyactive, and kill the MART-APL peptide pulsed T2 targets (FIG. 11a ).

Phenotypic Analysis of Immature DCs Maturing Under the PME-CD40L Process

DCs were matured on Day 5 with the PME-CD40L process described herein.Specifically monocytes were cultured in medium GM-CSF and IL-4 for 5days to produce immature CD83⁻ DCs. On day 5, the immature DCs were fedwith medium containing TNFα, IFNγ and PGE₂ (TIP). On day 6, the post TIPphenotype was determined (see Table IV). As shown in Table IV, themajority of cells were positive for CD80, CD83, CD86 and CD209. TheseDCs were also CCR7 negative (data not shown). The low percentage of CD14⁺ cells represent monocytes that did not differentiate into dendriticcells. On day 6, the CD83⁺ CCR7 ⁻ DCs were co-transfected (viaelectroporation) with 1 μg mRNA prepared from amplified renal cellcarcinoma RNA and 4 μg CD40L mRNA per million cells. CD40L expressionwas measured at 4 hours post transfection. The cells were cryopreservedin liquid nitrogen at 4 hrs post transfection. The post thaw recoveryand viability were measured immediately after thawing, and at 24 hourspost thawing. As can be seen, at 24 hours post thaw, the majority of DCsbecame CCR7+. The CCR7+ DCs were also positive for CD80, CD83 and CD86.The results of 3 separate runs are shown in Table IV.

TABLE IV Run data Run 1 Run 2 Run 3 Seeding density per flask 200 × 200× 200 × 10⁶ 10⁶ 10⁶ Number of flasks seeded 18 20 20 Post TIP Recovery(%) 8 24 15 Post TIP Viability (%) 97 95 93 Number of cuvettes 14 15(limited) 15 (limited) 4 hr post electroporation 64 43 73 Recovery (%) 4hr post electroporation 91 89 85 Viability (%) Number of vaccine doses13 9 15 from Run Post thaw Recovery (%) 86 94 85 Post thaw Viability (%)88 88 78 Predicted doses per 30 flasks 21 28 30 4 hr CD40L expression 8476 49 Post TIP DC phenotype % CD14 0.8 0.5 12 % CD80 100 100 98 % CD8399 92 82 % CD86 100 100 100 % CD209 98 99 100 mDC phenotype (post thaw)% CD14 3 0.3 1.4 % CD80 99 100 100 % CD83 100 100 98 % CD86 100 100 100% CD209 98 100 100 % CCR7 53 12 32 24 hr post thaw % CCR7 93 93 95 24 hrpost thaw ‘washout’ % viability 50 67 63 % recovery 36 46 73 24 hr postthaw transwell migration % Migration - media control 1.1 0.78 1.2 %Migration - 100 ng/ml 74 107 70 CCL19 and 21

DCs Matured via the PME-CD40L Process are Highly Migratory in Responseto Lymph Node Homing Chemokines, CCL19 and 21

PME-CD40L matured DCs were assayed for migration in response tochemokines, CCL19 and 21, twenty-four hours after co-transfection withtotal amplified RCC RNA and CD40L RNA. FIG. 12 shows that using fourindependent donors, that PME-CD40L matured DCs are highly migratory,consistent with the very high level of CCR7 expression achieved 24 hrspost electroporation with the PME-CD40L process (see Table IV).

DCs Matured via the PME-CD40L Process Show Significantly EnhancedImmunopotency over DCs Matured with the ‘CD40L Base Process’.

Despite the induction of primary immune responses by the ‘CD40L baseprocess’, the ‘post maturation electroporation-CD40L’ process, wherebyDCs are first matured with TNF-α, IFN-γ and PGE₂, prior toelectroporation with CD40L plus antigen-encoding mRNA, results in asignificant improvement in CTL activity using the MART antigen modelsystem. (FIG. 13). In addition, the PME-CD40L process was tested for theinduction of IFN-γ and IL-2 responses using fully autologous materialsderived from a renal cell carcinoma patient: patient DCs were preparedas described above for the PME-CD40L process, and electroporated withautologous total amplified RCC tumor RNA. The antigen loaded DCs werecultured with autologous patient CD8 T-cells, and the resultingresponder CTL were studied by intracellular cytokine staining inresponse to the eliciting DC, and to individual DCs transfected with thetumor-associated antigens, hTERT, Survivin and the RCC specific antigen,G250. DCs transfected with eGFP encoding mRNA were used as negativecontrol stimulators. FIG. 14 shows that patient T-cells responded to thetotal amplified RCC RNA loaded DCs, and also to the threetumor-associated antigens, with both IFN-γ and IL-2 frequencies higherthan that induced by the eGFP mRNA transfected negative control.(Response to eGFP subtracted from total response to each RCC associatedDC target)

DCs Matured by the ‘base CD40L Process’ and Pulsed with KRN7000 CanRecruit NKT-Cells which Enhance the Induction of primary CTLs.

MART-1 mRNA-loaded, CD40L base process matured DC, pulsed with KRN7000,increase the frequency of NKT-cells in PBMC cultures versus the samemature RNA loaded DCs pulsed with vehicle in place of KRN7000, asdefined by CD1d/KRN7000-tetramer staining (FIG. 15a ). Using tetrameranalysis for responder CTL (MART-1/HLA-A2), the presence of KRN7000pulsed onto MART-1 mRNA transfected DC significantly increases thefrequency of MART reactive T-cells (FIG. 15b ). Thus, the expansion ofNKT-cells in the PBMC cultures provides an amplification loop, probablyachieved by NKT-cell derived ‘help’, that can support primary CD8 CTLdevelopment.

Optimization of CD40L mRNA

The CD40L RNA used in the original DC experiments demonstrating apreferred way of maturation was transcribed from plasmid template pCR2.1CD40L WT. The preferred CD40L RNA contains an ARCA cap analog and polyAtail. The plasmid pCR2.1 CD40L WT was modified by removal of anXbaI-EcoRV fragment located 5′ of the initiator ATG codon. The fragmentencompassed 32 nucleotides of vector sequence and contained threecryptic potential initiator ATG codons. The rationale for thismodification was that these additional ATG's might interfere withefficient initiation of CD40L translation by competing with the accurateCD40L translation initiation site. The coding sequence of CD40L remainedunaffected by these modifications. CD40L RNA transcribed from themodified plasmid template performed better than the current CD40Lreference standard (pCR2.1 CD40L WT) in two independent DC transfectionexperiments as measured by induction of IL-12 expression. The modifiedplasmid is referred to as pCR2.1 CD40L WT Delta X-E.

In addition we wished to determine whether expression of the CD40L RNAcan be further optimized by placing the CD40L 5′ untranslated regiondirectly upstream of the CD40L initiator codon. The pCR2.1 CD40L WTDelta X-E plasmid was further modified by the insertion of 39 by CD40L5′ untranslated sequence located immediately upstream of the CD40Ltranslation start site, resulting in the construct pCR2.1 CD40L+5′UTR.RNA transcribed from this plasmid (pCR2.1 CD40L+5′UTR) did not performas well as the RNA described from CD40L WT Delta X-E but rather,performed similarly to the current CD40L transcribed from pCR2.1 CD40LWT (FIGS. 18 and 19). The DNA sequence corresponding to the CD40L RNAtranscribed from the pCR2.1 CD40L WT Delta X-E plasmid is shown in SEQID NO: 13. The ATG start codon begins at position 41 of SEQ ID NO: 13.

Short Isoform of CD40L Protein

One microgram of each of the CD40L RNAs described below was translatedin vitro using Wheat Germ extract (Promega) in the presence of tracer³⁵S-labeled Methionine. 5 μL of each translation mixture was resolved bySDS-PAGE electrophoresis and transferred to a nylon membrane. Themembrane was exposed to a Phosphoimager screen and scanned using theStorm Imager (Amersham). The results are shown in FIG. 20. Lanes 1 and 2represent the in vitro translated products from the pCR2.1 CD40L WT mRNA(WT), uncapped, and capped, respectively. Lanes 3 and 4-5 represent thein vitro translated products from the pCR2.1 CD40L WT Delta X-E mRNA(ΔE), uncapped, and capped, respectively. Lane 6 represents the in vitrotranslated product of the capped pCR2.1 CD40L+5′UTR mRNA. Examination ofthe radiolabeled translation products reveals that some RNAs give riseto two major proteins. Sequence analysis of the CD40L coding regionreveals that more than one in-frame methionine residue within the codingregion can give rise to a partial CD40L protein sequence (see SEQ ID NO:2). Since the truncated protein will be in frame it will still stainpositive with anti-CD154 antibodies. Analysis of the in vitro translatedproduct from the construct containing the naturally occurring CD40L5′UTR (pCR2.1 CD40L+5′UTR) encodes only one CD40L protein (FIG. 20).However this +5′UTR CD40L construct also exhibits the lowest IL-12potentiation (FIG. 19). In contrast, both the WT and Delta XE constructsappear to produce a shorter protein product in approximately equalproportion with the full length product and exhibit the highest IL-12inducing capacity.

We hypothesized that the lower molecular weight protein productinitiates from an internal methionine and could be a more active form ofCD40L for induction of IL-12 cytokine. This hypothesis was tested in thenext set of experiments by removing the most amino-terminal methioninecodon by site directed mutagenesis of the ATG start codon to GCG, sothat translation would begin at the second internal methionine of thewild type CD40L protein. This construct was called CD40L ΔXE-MET#1(CD40L ΔXE minus MET#1). The mRNA transcribed from this plasmid is shownin SEQ ID NO: 30. SEQ ID NO: 30 encodes the polypeptide of SEQ ID NO:31, which is equivalent to amino acid residues 21-261 of SEQ ID NO: 2.In addition, we tested another construct where translation initiationfrom the first initiator methionine is enhanced by optimizing the ATGcodon with a consensus Kozak sequence. Both modifications were made inthe Delta XE plasmid background as this plasmid template consistentlyencodes a more active CD40L RNA than does the WT plasmid. A new lot ofunmodified Delta XE RNA was made for use in this assay as a control. TheRNAs from these constructs were produced, polyadenylated and purified.The RNAs were translated in vitro in the presence of ³⁵S-labeledmethionine and analyzed by SDS-PAGE. As shown in FIG. 21, we confirmedthe original observation that two isoforms of CD40L protein are madefrom the WT and Delta XE CD40L RNAs. The CD40L WT RNA produces twoisoforms with the lower isoform slightly exceeding 50% of the totalprotein produced (FIG. 21, lane 1). Delta XE (second lot of RNA) encodesthe shorter form in a slightly higher ratio, while the CD40L RNA withthe naturally occurring 5′UTR gives rise to predominantly longer form(FIG. 21, lanes 2 and 5). As predicted, the new RNA with an optimizedKozak sequence surrounding the first ATG gave rise to predominantlylonger form (75% of total) (FIG. 21, lane 4). Most importantly, theCD40L RNA lacking the first methionine resulted in exclusively the shortform of the protein (FIG. 21, lane 6). No difference was noticed inprotein produced from RNA capped co-transcriptionally (FIG. 21, lane 2)versus RNA capped post-transcriptionally using capping enzyme (ΔE (enz),FIG. 21, lane 3).

We next evaluated the amount of CD40L (CD154) protein produced from eachconstruct in transfected DCs. The percentage of CD40L-positive cells(FIG. 22, left panel) is roughly equivalent in all conditions tested,indicating that transfection efficiency for each RNA is similar. Themean fluorescent intensity (FIG. 22, right panel) is proportional to theamount of CD40L protein produced in the cell. The ΔE RNA in this assayappears to perform better than the wild type control, as we haveconsistently observed. The Delta ΔE+Kozak RNA which, encodespredominantly the larger CD40L isoform surprisingly produced the lowestamount of CD40L protein, while the ΔXE-Met#1 RNA, which encodesexclusively the shorter isoform in vitro, produces the highest CD40Lprotein levels in transfected DCs.

The levels of CD40L protein expression as well as relative ratio oflong/short CD40L protein isoform were measured in order to determinewhether there was a correlation with IL-12 secretion levels. FIG. 23shows that the levels of IL-12 expression in this assay correlated withmean fluorescent intensity of cells stained with anti-CD154 antibodies.A closer look at the same data is presented in a FIG. 24. The absoluteamounts of IL-12 expression in the DC transfection assay areproportional to the amount of the short CD40L protein isoform producedin the in vitro translation assay. The addition of the 3′UTR ofrotavirus gene 6 to the ΔXE construct (to produce the Rot6 3′UTR ΔEplasmid) resulted in IL-10 and IL-12 expression levels similar to thoseobserved for ΔXE-Met#1 (FIG. 25). The sequence of the mRNA transcribedfrom the Rot6 3′UTR ΔE plasmid is shown in SEQ ID NO: 32. The cDNAencoding the RNA transcribed from the ΔXE-Met#1 Rotavirus gene 6 3′UTRconstruct is shown in SEQ ID NO: 33.

It was assumed that translation of the ΔXE-Met#1 RNA initiated at thesecond ATG codon of the CD40L CDS, to produce an N-terminal CD40Ltruncated protein beginning at the second internal methionine of theCD40L (i.e., amino acid residue 21 of SEQ ID NO: 2). In order to confirmthis assumption, the ΔE construct was subjected to site directedmutagenesis to alter either the first two methionine codons(ΔXE-Met#1,2), the first three methionine codons (ΔXE-Met#1-3); or thefirst four methionine codons (ΔXE-Met#1-4). The in vitro translationproducts produced by these construct are shown in FIG. 26. Secretionlevels of IL-10 and IL-12 by dendritic cells transfected with thesemodified CD40L RNAs are shown in FIG. 27. Deletion of the firstmethionine in the ΔXE-Met#1 construct results in high IL-12 secretionfrom DCs transfected with this RNA, while deletion of the first 2, 3 or4 methionines results in no IL-12 production. Therefore, transfection ofDCs with an RNA encoding the CD40L polypeptide of SEQ ID NO: 31 resultsin high levels of IL-12 expression. In contrast, CD40L polypeptidesinitiating at the 3^(rd), 4^(th) or 5^(th) internal ATG codon are notable to induce IL-12 secretion when they are expressed in DCs.

IL-10 and IL-12 Expression at 4 and 24 Hours Post-Transfection in DCsMatured by the PME-CD40L Process.

Immature DCs were phenotypically matured on Day 5 of culture with TNF-α(10 ng/ml), IFN-γ (1000 U/ml) and PGE₂ (1 μg/ml). On day 6, DCs wereharvested and electroporated with antigen and CD40L mRNA as describedabove, and cultured in X-VIVO 15 media containing 800 U/ml GM-CSF and500 U/ml IL-4 for 4 hours or 24 hours. Table V shows that supernatantsharvested 4 hours post-transfection (vaccine) produce little or noIL-12p70 or IL-10, while the levels increase at 24 hourspost-transfection.

TABLE V Cytokine secretion from PME-CD40L DCs IL-10 IL-12 pg/ml (s.d.)pg/ml (s.d.)  4 hours 0 (0)   17 (15.4) 24 hours 83.3 (26.3) 254.7(21.8) n = 3DCs matured via the PME-CD40L Process Secret Lower Levels of IL-12p70Compared to DCs Matured with the ‘CD40L Base Process’.

To compare IL-10 and IL-12 secretion levels, DCs were prepared using thestandard CD40L base process or PME-CD40L process, and secreted cytokinesin the culture medium were measured at 18-24 hours followingelectroporation. The results are shown in Table VI. In comparison to the‘CD40L base process’, the ‘post maturation electroporation-CD40L’process (i.e., whereby DCs are first matured with TNF-α, IFN-γ and PGE₂,prior to electroporation with CD40L plus antigen-encoding mRNA) resultsin lower levels of IL-12p70, while the levels of IL-10 are similar.However, DCs matured by either the CD40L base process, or the PME-CD40Lprocess secrete lower levels of IL-10 and higher levels of IL-12 ascompared to DCs matured by the cytokine cocktail process (IL-6, IL-1β,TNFα and PGE₂).

TABLE VI Cytokine secretion from PME-CD40L DCs DC maturation IL-10 IL-12process pg/ml (s.d.) pg/ml (s.d.) CD40L base process  89 (51) 1218 (86)PME-CD40L process 125 (64)  602 (53)

Cell Surface Staining and Measurement of Intracellular CytokineProduction

DCs were generated as described above, employing the PME-CD40L process,or DC electroporated with CD40L RNA and MART-1 RNA and cultured for 4hours with IFN-γ and PGE₂ or DC matured with TNFα, IFNγ, PGE₂ (TIP)cytokines overnight then electroported with Mart-1 RNA and cultured for4 hours or immature DC electroported with MART-1 RNA and co-culturedwith cytokine cocktail (IL-1β, IL-6, TNFα, IFNγ, PGE₂) for 4 hours DCand CD8 T cells were co-cultured as described for “CTL induction”. Onthe indicated day CTL were harvested and stimulated with T2 cellspreviously pulsed with 10 μg/ml of either the HLA-A201 restrictedMART-APL peptide (LAGIGILTV; SEQ ID NO: 24) or PSA-1 peptide(FLTPKKLQCV; SEQ ID NO: 26) by 1 hour incubation in FBS/RPMI media,washed and CTL were stimulated at a 10:1 ratio with T2 cells. At the 1hour time point brefeldin A was added and cultures were allowed toincubate for an additional 3 hours. CTL were then surfaced stained withantibodies to CD8 receptor and MART-1/A2 pentamers to detect thefrequency of antigen specific CTL. CTL were then permiablized withSaponin buffer to detect intracellular production of IL-2 and IFN-γusing cytokine specific antibodies. In some cases CTL were harvestedfrom co-cultures and surfaced stained with monoclonal antibodies to theCD8 receptor, CD28 receptor, CD45RA molecule and MART-1/A2 pentamers.

DCs Matured via the PME-CD40L Process Shown Significantly EnhancedImmunopotency over DCs Matured with the ‘CD40L Base Process’ and OtherProcesses of Inducing Maturation of DCs

FIG. 28 shows the increased percentage of Mart-1 reactive CTL on day 25in co-cultures with DC generated with the PME-CD40L process compared toother methods of generating DC such as DC electroporated with CD40L RNAand Mart-1 RNA and cultured for 4 hours with IFN-γ and PGE₂ (CD40L) orDC matured with cytokines (TNFα, IFN-γ and PGE₂) overnight thenelectroported with Mart-1 RNA and cultured for 4 hours (TIP) or immatureDC electroported with MART-1 RNA and co-cultured with cytokine cocktail(IL-6, IL-1β, TNFα, IFNγ, PGE₂) for 4 hours (Cytokines). MART-1 specificCTL were identified by co-staining with MART-1/HLA-A2 pentamers andanti-CD8 receptor antibodies. FIG. 28 (bottom panels) and FIG. 29 showthat the majority of MART-1 CTL generated in the presence of PME-CD40LDC express the CD28 receptor in contrast to CTL generated in thepresence of other DC preparations.

The time course of CD28 receptor expression depicted in FIG. 29 showsthat as early as day 14 during the co-culture period, 89% of MART-1 CTLfrom PME-CD40L DC co-cultures express the CD28 receptor. Moreover CTLmaintain CD28 receptor expression throughout the co-culture period. Thisis in contrast to both the TIP DC and cytokine DC co-cultures where CD28expression declines over time. CD28 receptor positive cells areconsidered antigen experienced MART-1 CTL based on the lack of specificstaining with antibody to the CD45RA molecule.

FIG. 30 shows that PME-CD40L were able to induce the greatest number ofCTL producing IFN-γ^(HI)/IL-2^(HI) (60%) compare to the other DCprocesses TIP (6.5%), CD40L (50%), and Cytokines (14%) on day 10 ofculture.

FIG. 31 shows the mean fluorescence intensity (MFI) of IFN-γ positiveCTL as a measure of the overall level of cytokine being produced byMart-1CTL. The highest level of IFN-γ production is seen in Mart-1 CTLPME-CD40L DC co-cultures.

DCs Matured by the PME-CD40L Process Preferentially Induce a Populationof Antigen-Specific Effector/Memory CTL

Despite the induction of primary immune responses by the ‘CD40L baseprocess’, the ‘PME-CD40L’ process, whereby DCs are first matured withTNF-α, IFN-γ and PGE₂, prior to electroporation with CD40L plusantigen-encoding mRNA, results in a significant improvement in CTLactivity using the MART antigen model system. (FIG. 13). Furtheranalysis of these antigen reactive CTL revealed that DC matured via thePME-CD40L process induces a greater number of MART-1 reactive CTL thanDC matured using other methods as described in FIG. 28. Furthermore theantigen experienced MART-1 reactive CTL continue to maintain expressionof the CD28 receptor in contrast to MART-1 CTL assayed from the other DCco-cultures (FIGS. 28 and 29). These cells are defined as antigenexperienced CTL by the lack of the CD45RA molecule expression (Tomiyamaet al. J. Immunol. (2002) 168:5538-5550), but are not consideredterminally differentiated effector CTL based on the expression of CD28receptor and their increased numbers present in PME-CD40L DCco-cultures. These CTL differ from other effector/memory CTL that havebeen reported in the literature where viral specific CTL are CD28negative and proliferate poorly (Weekes et al. J. Immunol. (1999)162:7569-7577). PME-CD40L DC induce a novel population of antigenspecific CTL that retain the capacity to proliferate in the presence ofantigen. Therefore these CTL differ from the type of CTL generated withother methods of generating DC. This is the first report of a dendriticcell that preferentially induces a population of CD28⁺ CD45RA⁻memory/effector T cells from a population of naïve T cells orantigen-specific T cells.

During chronic antigen stimulation similar to what is seen in certainviral infections such as CMV (Topp et al. J Exp Med (2003) 198:947-955)and HIV (Lieberman et al. 2001 Blood 98:1667-1677) there is a loss ofexpression of CD28 and a loss of the ability to produce IL-2. FIG. 30shows that PME-CD40L generated DC in contrast to other methods ofgenerating mature DC are capable of priming MART-1 specific CTL thatretain the capacity to produce both IL-2 and IFN-γ. Where the PME-CD40LDC were able to induce the highest percentage of IFN-g/IL-2 doublepositive CTL (60%) compare to the other DC processes TIP (6.5%), CD40L(50%), and Cytokines (14%). It has been reported that loss of CD28receptor on HIV specific CTL parallels progressive HIV viral replication(Gamberg et al. Immunology and Cell Biology (2004) 82:38-46). WhereasHIV specific CTL that produce IFNg/IL-2 are able to support theproliferation of HIV specific CTL (Zimmerli et al. PNAS (2005)102:7239-7244). While these IFNg/IL-2 producing CTL have a CD45RAnegative effector phenotype they were not characterized using the CD28receptor. Topp et al. (J Exp Med (2003) 198:947-955) showed that byre-introducing the CD28 receptor into a CD28 negative CMV or HIVspecific CTL could restore IL-2 production and sustain CTLproliferation. The PME-CD40L process of generating DC unlike othermethods of generating DC are capable of inducing antigen specific CTLthat are CD28 positive and retain the capacity to produce high levels ofIL-2 and IFN-γ. Thus the PME-CD40L process of generating DC is capableof supporting long term antigen specific CTL effector function andinducing a preferred phenotype of effector memory CTL that retains thecapacity to expand, produce cytokines and kill target cells all criticalevents mediating robust long-term CTL effector function.

What is claimed is:
 1. A method for preparing mature dendritic cells(DCs), comprising the sequential steps of: (a) signaling isolatedimmature dendritic cells (iDCs) with a first signal comprising aninterferon gamma receptor (IFN-γR) agonist, and optionally, a TNF-αRagonist, to produce IFN-γR agonist signaled dendritic cells; and (b)signaling said IFN-γR agonist signaled dendritic cells with a secondtransient signal comprising an effective amount of a CD40 agonist toproduce CCR7⁺ mature dendritic cells.